LAG-3 TARGETED HETERODIMERIC FUSION PROTEINS CONTAINING IL-15/IL-15RA Fc-FUSION PROTEINS AND LAG-3 ANTIGEN BINDING DOMAINS

ABSTRACT

The present invention is directed to novel targeted heterodimeric fusion proteins comprising an IL-15/IL-15Rα Fc-fusion protein and a LAG-3 antibody fragment-Fc fusion protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNos. 62/659,624 filed Apr. 18, 2018 and 62/783,107, filed Dec. 20, 2018,which are expressly incorporated herein by reference in their entirety,with particular reference to the figures, legends, and claims therein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on May 31, 2019, is named067461-5222-US_SL.txt and is 421,438 bytes in size.

BACKGROUND OF THE INVENTION

Two very promising approaches in cancer immunotherapy includecytokine-based treatments and blockade of immune checkpoint proteinssuch as PD-1.

Cytokines such as IL-2 and IL-15 function in aiding the proliferationand differentiation of B cells, T cells, and NK cells. Both cytokinesexert their cell signaling function through binding to a trimericcomplex consisting of two shared receptors, the common gamma chain (γc;CD132) and IL-2 receptor beta-chain (IL-2R1; CD122), as well as an alphachain receptor unique to each cytokine: IL-2 receptor alpha (IL-2Rα;CD25) or IL-15 receptor alpha (IL-15Rα; CD215). Both cytokines areconsidered as potentially valuable therapeutics in oncology, and IL-2has been approved for use in patients with metastatic renal-cellcarcinoma and malignant melanoma. Currently, there are no approved usesof recombinant IL-15, although several clinical trials are ongoing.However, as potential drugs, both cytokines suffer from a very fastclearance, with half-lives measured in minutes. IL-2 immunotherapy hasbeen associated with systemic toxicity when administered in high dosesto overcome fast clearance. Such systemic toxicity has also beenreported with IL-15 immunotherapy in recent clinical trials (Guo et al.,J Immunol, 2015, 195(5):2353-64).

Immune checkpoint proteins such as PD-1 are up-regulated following Tcell activation to preclude autoimmunity by exhausting activated T cellsupon binding to immune checkpoint ligands such as PD-L1. However, immunecheckpoint proteins are also up-regulated in tumor-infiltratinglymphocytes (TILs), and immune checkpoint ligands are overexpressed ontumor cells, contributing to immune escape by tumor cells. De-repressionof TILs by blockade of immune checkpoint interactions by drugs such asOpdivo® (nivolumab) and Keytruda® (pembrolizumab) have proven highlyeffective in treatment of cancer. Despite the promise of checkpointblockade therapies such as nivolumab and pembrolizumab, many patientsstill fail to achieve sufficient response to checkpoint blockade alone.

Therefore, there remains an unmet need in oncology treatment fortherapeutic strategies with cytokines that do not require high doses andare targeted to tumors to avoid systemic toxicity. Further, there is aneed to identify additional therapeutic modalities to stack withcheckpoint blockade that could increase patient response rate.

To address these needs and caveats, provided herein are novelLAG-3-targeted IL-15 heterodimeric fusion proteins with enhancedhalf-life and more selective targeted of TILs to improve safety profile,and which synergistically combine with checkpoint blockade antibodies(FIG. 1).

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a targeted IL-15/IL-15Rαheterodimeric protein comprising: (a) a first monomer comprising, fromN-to C-terminal: i) an IL-15 sushi domain; ii) a first domain linker;iii) a variant IL-15 domain; iv) a second domain linker; v) a firstvariant Fc domain comprising CH2-CH3; and (b) a second monomercomprising, from N-to C-terminal: i) a scFv domain; ii) a third domainlinker; iii) a second variant Fc domain comprising CH2-CH3; wherein thescFv domain comprises a first variable heavy domain, an scFv linker anda first variable light domain, wherein the scFv domain binds humanLAG-3.

In other aspects of the present invention, provided herein is a targetedIL-15/IL-15Rα heterodimeric protein comprising: (a) a first monomercomprising, from N-to C-terminal: i) an IL-15 sushi domain; ii) a firstdomain linker; iii) a first variant Fc domain comprising CH2-CH3; (b) asecond monomer comprising, from N-to C-terminal: i) a scFv domain; ii) athird domain linker; iii) a second variant Fc domain comprising CH2-CH3;wherein the scFv domain comprises a first variable heavy domain, an scFvlinker and a first variable light domain; and (c) a third monomercomprising a variant IL-15 domain; wherein the scFv domain binds humanLAG-3.

In one aspect, provided are “scIL-15/Rα X Fab” format heterodimericproteins. Such “scIL-15/Rα X Fab” format heterodimeric proteins include:a) a first monomer comprising, from N-to C-terminal: i) anIL-15Rα(sushi) domain; ii) a first domain linker; iii) an IL-15 variant;iv) a second domain linker; v) a first variant Fc domain comprisingCH2-CH3; b) a second monomer comprising, from N-to C-terminal,VH-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a second variant Fc domain; andc) a third monomer comprising a VL-CL. The VH and VL are a variableheavy domain and a variable light domain, respectively, that form ahuman LAG-3 antigen binding domain. In some embodiments, the seconddomain linker is an antibody hinge.

In certain embodiments of the “scIL-15/Rα X Fab” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU numbering.In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment of the “scIL-15/Rα X Fab” formatheterodimeric protein, the “scIL-15/Rα X Fab” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal:i) an IL-15Rα(sushi) domain; ii) a first domain linker; iii) an IL-15variant; iv) a hinge; v) a first variant Fc domain comprising CH2-CH3;b) a second monomer comprising, from N-to C-terminal,VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a second variant Fc domain;and c) a third monomer comprising a VL-CL. The VH and VL are a variableheavy domain and a variable light domain, respectively, that form ahuman LAG-3 antigen binding domain. In such embodiments, the firstvariant Fc domain comprises skew variants L368D/K370S and the secondvariant Fc domain comprises skew variants S364K/E357Q the first andsecond variant Fc domains each comprise FcKO variantsE233P/L234V/L235A/G236del/S267K, the first variant Fc domain comprisespI variants Q295E/N384D/Q418E/N421D, wherein numbering is according toEU numbering. In some embodiments, the hinge of the first monomercomprises amino acid substitution C220S, wherein numbering is accordingto EU numbering. In an exemplary embodiment, the first and secondvariant Fc domains each further comprise half-life extension variantsM428:/N434S.

In some embodiments of the “scIL-15/Rα X Fab” format heterodimericprotein, the IL-15 variant of the heterodimeric protein provided hereincomprises an amino acid substitution(s) selected from the groupconsisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E, N4D/N65D,D30N/N65D, and D30N/E64Q/N65D. In an exemplary embodiment, the IL-15variant comprises amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D

In an exemplary embodiment, the “scIL-15/Rα X Fab” format heterodimericprotein is XENP27972, XENP27973, XENP27977, XENP27978, XENP029486,XENP029487, XENC1000, XENC1001, XENC1002, XENC1003, XENC1004 orXENC1005.

In certain embodiment, the VH and VL of the scIL-15/Rα X Fab” formatheterodimeric proteins provided herein are the variable heavy domain andvariable domain of any of the LAG-3 antigen binding domains in FIGS. 12and 13. In an exemplary embodiment, the LAG-3 antigen binding domain is2A11_H1.144_L2.142 and 7G8_H3.30_L1.34.

In one aspect, provided herein is a heterodimeric protein having the“scIL-15/Rα X scFv” format. In one embodiment, the heterodimeric proteinincludes: a) a first monomer comprising, from N-to C-terminal: i) anIL-15Rα(sushi) domain; ii) a first domain linker; iii) an IL-15 variant;iv) a second domain linker; and v) a first variant Fc domain comprisingCH2-CH3; and b) a second monomer comprising, from N-to C-terminal: i) ascFv domain; ii) a third domain linker; and iii) a second variant Fcdomain comprising CH2-CH3. In some embodiments, the scFv domaincomprises a variable heavy domain (VH), an scFv linker and a variablelight domain (VL), and the scFv domain binds human LAG-3. In someembodiments of the “scIL-15/Rα X scFv” format heterodimeric protein, thesecond domain linker and the third domain linker are each an antibodyhinge.

In certain embodiments, the first variant Fc domain the second variantFc domain comprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In some embodiments of the “scIL-15/Rα X scFv” format, the heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal:i) an IL-15Rα(sushi) domain; ii) a first domain linker; iii) an IL-15variant; iv) a hinge; and v) a first variant Fc domain comprisingCH2-CH3; and b) a second monomer comprising, from N-to C-terminal: i) ascFv domain; ii) a hinge; and iii) a second variant Fc domain comprisingCH2-CH3. In some embodiments, the scFv domain comprises a variable heavydomain (VH), an scFv linker and a variable light domain (VL), and thescFv domain binds human LAG-3. In such embodiments, the first variant Fcdomain comprises skew variants L368D/K370S and the second variant Fcdomain comprises skew variants S364K/E357Q, the first and second variantFc domains each comprise FcKO variants E233P/L234V/L235A/G236del/S267K,the first variant Fc domain comprises pI variantsQ295E/N384D/Q418E/N421D, and the numbering is according to EU numbering.In certain embodiments, the first and second hinges each comprise aminoacid substitution C220S, wherein numbering is according to EU numbering.In one embodiment, the first and second variant Fc domains each furthercomprise half-life extension variants M428:/N434S.

In another aspect, provided herein are “scFv X ncIL-15/Rα” formatheterodimeric proteins. Such heterodimeric proteins include: a) a firstmonomer comprising, from N-to C-terminal: i) a scFv domain; ii) a firstdomain linker; and iii) a first variant Fc domain comprising CH2-CH3; b)a second monomer comprising, from N-to C-terminal: i) an IL-15Rα(sushi)domain; ii) a second domain linker; and iii) a second variant Fc domaincomprising CH2-CH3; and c) a third monomer comprising an IL-15 variant.The scFv domain comprises a variable heavy domain (VH), an scFv linkerand a variable light domain (VL), and the scFv domain binds human LAG-3.In one embodiment, the first domain linker and the second domain linkerare each an antibody hinge.

In some embodiments of the “scFv X ncIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment, the “scFv X ncIL-15/Rα” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal:i) a scFv domain; ii) a hinge; and iii) a first variant Fc domaincomprising CH2-CH3; b) a second monomer comprising, from N-toC-terminal: i) an IL-15Rα(sushi) domain; ii) a hinge; and iii) a secondvariant Fc domain comprising CH2-CH3; and c) a third monomer comprisingan IL-15 variant. Further, the scFv domain comprises a variable heavydomain (VH), an scFv linker and a variable light domain (VL), and thescFv domain binds human LAG-3. In such embodiments, the first variant Fcdomain comprises skew variants L368D/K370S and the second variant Fcdomain comprises skew variants S364K/E357Q, the first and second variantFc domains each comprise FcKO variants E233P/L234V/L235A/G236del/S267K,the first variant Fc domain comprises pI variantsQ295E/N384D/Q418E/N421D, and wherein numbering is according to EUnumbering. In certain embodiments, the first and second hinges eachcomprise amino acid substitution C220S, wherein numbering is accordingto EU numbering. In one embodiment, the first and second variant Fcdomains each further comprise half-life extension variants M428:/N434S.

In another aspect, provided herein are “scFv x dsIL-15/Rα” formatheterodimeric proteins. The “scFv x dsIL-15/Rα” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal:i) a variant IL-15Rα(sushi) domain comprising an amino acid substitutedfor a cysteine residue; ii) a first domain linker; and iii) a firstvariant Fc domain comprising CH2-CH3; b) a second monomer comprising,from N-to C-terminal: i) a scFv domain; ii) a second domain linker; iii)a second variant Fc domain comprising CH2-CH3; an c) a third monomercomprising an IL-15 variant comprising an amino acid substituted for acysteine residue. The scFv domain comprises a variable heavy domain(VH), an scFv linker and a variable light domain (VL), wherein thecysteine residue of the variant IL-15Rα(sushi) domain and the cysteineresidue of the IL-15 variant form a disulfide bond and the scFv domainbinds human LAG-3. In certain embodiments, the first domain linker andthe second domain linker are each an antibody hinge.

In some embodiments of the “scFv x dsIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment, the “scFv x dsIL-15/Rα” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal:i) a variant IL-15Rα(sushi) domain comprising an amino acid substitutedfor a cysteine residue; ii) a hinge; and iii) a first variant Fc domaincomprising CH2-CH3; b) a second monomer comprising, from N-toC-terminal: i) a scFv domain; ii) a hinge; iii) a second variant Fcdomain comprising CH2-CH3; an c) a third monomer comprising an IL-15variant comprising an amino acid substituted for a cysteine residue. ThescFv domain comprises a variable heavy domain (VH), an scFv linker and avariable light domain (VL), wherein the cysteine residue of the variantIL-15Rα(sushi) domain and the cysteine residue of the IL-15 variant forma disulfide bond and the scFv domain binds human LAG-3. In suchembodiments, the first variant Fc domain comprises skew variantsL368D/K370S and the second variant Fc domain comprises skew variantsS364K/E357Q, the first and second variant Fc domains each comprise FcKOvariants E233P/L234V/L235A/G236del/S267K, the first variant Fc domaincomprises pI variants Q295E/N384D/Q418E/N421D, wherein numbering isaccording to EU numbering. In certain embodiments, the hinges of thefirst and second monomers each comprise amino acid substitution C220S,wherein numbering is according to EU numbering. In some embodiments, thefirst and second variant Fc domains each comprise half-life extensionvariants M428:/N434S.

In one aspect, provided herein are “Fab X ncIL-15/Rα” formatheterodimeric proteins. Such heterodimeric proteins include: a) a firstmonomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, whereinthe CH2-CH3 is a first variant Fc domain; b) a second monomercomprising, from N-to C-terminal: i) an IL-15Rα(sushi) domain; ii) afirst domain linker; iii) a first variant Fc domain comprising CH2-CH3;c) a third monomer comprising a light chain comprising VL-CL; and d) afourth monomer comprising an IL-15 variant. The VH and VL are a variableheavy domain and a variable light domain, respectively, that form ahuman LAG-3 antigen binding domain. In some embodiments, the firstdomain linker is an antibody hinge.

In some embodiments of the “Fab X ncIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU numbering.In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In exemplary embodiments, the “Fab X ncIL-15/Rα” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal,VH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a first variant Fc domain;b) a second monomer comprising, from N-to C-terminal: i) anIL-15Rα(sushi) domain; ii) a hinge; iii) a first variant Fc domaincomprising CH2-CH3; c) a third monomer comprising a light chaincomprising VL-CL; and d) a fourth monomer comprising an IL-15 variant.The VH and VL are a variable heavy domain and a variable light domain,respectively, that form a human LAG-3 antigen binding domain. In suchembodiments, the first variant Fc domain comprises skew variantsL368D/K370S and the second variant Fc domain comprises skew variantsS364K/E357Q, the first and second variant Fc domains each comprise FcKOvariants E233P/L234V/L235A/G236del/S267K, and the hinge-first variant Fcdomain of the first monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In some embodiments, the hinge of the second monomercomprises amino acid substitution C220S, wherein numbering is accordingto EU numbering. In certain embodiments, the first and second variant Fcdomains each further comprise half-life extension variants M428:/N434S.

In another aspect, provided herein are “Fab X dsIL-15/Rα” formatheterodimeric proteins. Such “Fab X dsIL-15/Rα” format heterodimericproteins include: a) a first monomer comprising, from N-to C-terminal,VH-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first variant Fc domain; b) asecond monomer comprising, from N-to C-terminal: i) a variantIL-15Rα(sushi) domain comprising an amino acid substituted for acysteine residue; ii) a first domain linker; and a first variant Fcdomain comprising CH2-CH3; c) a third monomer comprising, from N- toC-terminal, VL-CL; and d) a fourth monomer comprising an IL-15 variantcomprising an amino acid substituted for a cysteine residue. Further,the cysteine residue of the variant IL-15Rα(sushi) domain and thecysteine residue of the IL-15 variant form a disulfide bond, and the VHand VL are a variable heavy domain and a variable light domain,respectively, that form a human LAG-3 antigen binding domain. In someembodiments, the first domain linker is an antibody hinge.

In some embodiments of the “Fab X dsIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU numbering.In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment, the “Fab X dsIL-15/Rα” format heterodimericprotein includes: a) a first monomer comprising, from N-to C-terminal,VH-CH1-hinge-CH2-CH3, wherein CH2-CH3 is a first variant Fc domain; b) asecond monomer comprising, from N-to C-terminal: i) a variantIL-15Rα(sushi) domain comprising an amino acid substituted for acysteine residue; a hinge; and a first variant Fc domain comprisingCH2-CH3; c) a third monomer comprising, from N- to C-terminal, VL-CL;and d) a fourth monomer comprising an IL-15 variant comprising an aminoacid substituted for a cysteine residue. Further, the cysteine residueof the variant IL-15Rα(sushi) domain and the cysteine residue of theIL-15 variant form a disulfide bond, and the VH and VL are a variableheavy domain and a variable light domain, respectively, that form ahuman LAG-3 antigen binding domain. In such embodiments, the firstvariant Fc domain comprises skew variants L368D/K370S and the secondvariant Fc domain comprises skew variants S364K/E357Q the first andsecond variant Fc domains each comprise FcKO variantsE233P/L234V/L235A/G236del/S267K, and the hinge-first variant Fc domainof the first monomer comprises pI variantsN208D/Q295E/N384D/Q418E/N421D, wherein numbering is according to EUnumbering. In certain embodiments, the hinge of the second monomercomprises amino acid substitution C220S, wherein numbering is accordingto EU numbering. In some embodiments, the first and second variant Fcdomains each further comprise half-life extension variants M428:/N434S.

In one aspect, provided herein are “mAb-scIL-15/Rα” format heterodimericproteins. The “mAb-scIL-15/Rα” format heterodimeric proteins include: a)a first monomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3,wherein the CH2-CH3 is a first variant Fc domain; b) a second monomercomprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3-domainlinker-IL-15Rα(sushi) domain-domain linker-IL-15 variant, wherein theCH2-CH3 is a second variant Fc domain; and c) a third monomer and fourthmonomer that each comprises, from N- to C-terminal, VL-CL. Further, theVH of the first monomer and the VL of the third monomer form a firsthuman LAG-3 binding domain, and the VH of the second monomer and the VLof the fourth monomer form a second human LAG-3 binding domain.

In some embodiments of the “mAb-scIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU numbering.In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In some embodiments of the “mAb-scIL-15/Rα” format heterodimericprotein, the first variant Fc domain comprises skew variants L368D/K370Sand the second variant Fc domain comprises skew variants S364K/E357Q andthe first and second variant Fc domains each comprise FcKO variantsE233P/L234V/L235A/G236del/S267K, wherein numbering is according to EUnumbering. In certain embodiments, a) the hinge-first variant Fc domainof the first monomer further comprises pI substitutionsN208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc domain ofthe second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsN208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc domainof the second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K, wherein numbering is according to EUnumbering.

In some embodiments of the “mAb-scIL-15/Rα” format heterodimericprotein, the first variant Fc domain comprises skew variants S364K/E357Qand the second variant Fc domain comprises skew variants L368D/K370S,and the first and second variant Fc domains each comprise FcKO variantsE233P/L234V/L235A/G236del/S267K, wherein numbering is according to EUnumbering. In such embodiments, a) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsQ196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc domain ofthe second monomer further comprises pI variantsN208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsQ196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc domainof the second monomer further comprises pI variantsN208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EUnumbering.

In some embodiments of the “mAb-scIL-15/Rα” format heterodimericprotein, the first and second variant Fc domains each further comprisehalf-life extension variants M428:/N434S.

In another aspect, provided herein are “mAb-ncIL-15/Rα” formatheterodimeric proteins. Such heterodimeric protein include: a) a firstmonomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, whereinthe CH2-CH3 is a first variant Fc domain; b) a second monomercomprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3-domainlinker-IL-15Rα(sushi) domain, wherein the CH2-CH3 is a second variant Fcdomain; c) a third monomer comprising an IL-15 variant; and d) a fourthand fifth monomer that each comprises, from N- to C-terminal, VL-CL. TheVH of the first monomer and the VL of the fourth monomer form a firsthuman LAG-3 binding domain, and the VH of the second monomer and the VLof the fifth monomer form a second human LAG-3 binding domain.

In some embodiments of the “mAb-ncIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment of the “mAb-ncIL-15/Rα” format heterodimericprotein, the first variant Fc domain comprises skew variants L368D/K370Sand the second variant Fc domain comprises skew variants S364K/E357Q,and the first and second variant Fc domains each comprise FcKO variantsE233P/L234V/L235A/G236del/S267K, wherein numbering is according to EUnumbering. In some embodiments, a) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsN208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc domain ofthe second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsN208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc domainof the second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K, wherein numbering is according to EUnumbering.

In another exemplary embodiment of the “mAb-ncIL-15/Rα” formatheterodimeric protein, the first variant Fc domain comprises skewvariants S364K/E357Q and the second variant Fc domain comprises skewvariants L368D/K370S, and the first and second variant Fc domains eachcomprise FcKO variants E233P/L234V/L235A/G236del/S267K, whereinnumbering is according to EU numbering. In certain embodiments, a) thehinge-first variant Fc domain of the first monomer further comprises pIsubstitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second variantFc domain of the second monomer further comprises pI variantsN208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsQ196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc domainof the second monomer comprises pI variantsN208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EUnumbering.

In certain embodiments, the first and second variant Fc domains eachfurther comprise half-life extension variants M428:/N434S.

In another aspect, provided herein are “mAb-dsIL-15/Rα” heterodimericproteins. Such “mAb-dsIL-15/Rα” heterodimeric proteins include: a) afirst monomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3,wherein the CH2-CH3 is a first variant Fc domain; b) a second monomercomprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3-domainlinker-variant IL-15Rα(sushi) domain, wherein the variant IL-15Rα(sushi)domain an amino acid substituted for a cysteine residue and wherein theCH2-CH3 is a second variant Fc domain; c) a third monomer comprising anIL-15 variant comprising an amino acid substituted for a cysteineresidue; and d) a fourth and fifth monomer that each comprises, from N-to C-terminal, VL-CL. The cysteine residue of the variant IL-15Rα(sushi)domain and the cysteine residue of the IL-15 variant form a disulfidebond, the VH of the first monomer and the VL of the fourth monomer forma first human LAG-3 binding domain, and the VH of the second monomer andthe VL of the fifth monomer form a second human LAG-3 binding domain.

In some embodiments, the first variant Fc domain the second variant Fcdomain comprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q according to EU numbering.In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment of the “mAb-dsIL-15/Rα” heterodimericproteins, the first variant Fc domain comprises skew variantsL368D/K370S and the second variant Fc domain comprises skew variantsS364K/E357Q and the first and second variant Fc domains each compriseFcKO variants E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering. In some embodiments, a) the hinge-firstvariant Fc domain of the first monomer further comprises pIsubstitutions N208D/Q295E/N384D/Q418D/N421D and the hinge-second variantFc domain of the second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsN208D/Q295E/N384D/Q418D/N421D; or c) the hinge-second variant Fc domainof the second monomer further comprises pI variantsQ196K/I199T/P271R/P228R/N276K, wherein numbering is according to EUnumbering.

In another exemplary embodiment of the “mAb-dsIL-15/Rα” heterodimericproteins, the first variant Fc domain comprises skew variantsS364K/E357Q and the second variant Fc domain comprises skew variantsL368D/K370S, and the first and second variant Fc domains each compriseFcKO variants E233P/L234V/L235A/G236del/S267K, wherein numbering isaccording to EU numbering. In certain embodiments, a) the hinge-firstvariant Fc domain of the first monomer further comprises pIsubstitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second variantFc domain of the second monomer further comprises pI variantsN208D/Q295E/N384D/Q418D/N421D; b) the hinge-first variant Fc domain ofthe first monomer further comprises pI substitutionsQ196K/I199T/P271R/P228R/N276K; or c) the hinge-second variant Fc domainof the second monomer further comprises pI variantsN208D/Q295E/N384D/Q418D/N421D, wherein numbering is according to EUnumbering. In certain embodiments, the first and second variant Fcdomains each further comprise half-life extension variants M428:/N434S.

In one aspect, provided herein are “central-IL-15/Rα” formatheterodimeric proteins. Such “central-IL-15/Rα” format heterodimericproteins include: a) a first monomer comprising, from N- to C-terminal,a VH-CH1-domain linker-IL-15 variant-hinge-CH2-CH3, wherein the CH2-CH3is a first variant Fc domain; b) a second monomer comprising, from N- toC-terminal, a VH-CH1-domain linker-IL-15Rα(sushi) domain-hinge-CH2-CH3,wherein the CH2-CH3 is a second variant Fc domain; and c) a third andfourth monomer that each comprises, from N- to C-terminal, VL-CL. The VHof the first monomer and the VL of the third monomer form a first humanLAG-3 binding domain, and the VH of the second monomer and the VL of thefourth monomer form a second human LAG-3 binding domain.

In some embodiments of the “central-IL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment, the first variant Fc domain comprises skewvariants L368D/K370S and the second variant Fc domain comprise the skewvariant pair S364K/E357Q, the first and second variant Fc domains eachcomprise FcKO variants E233P/L234V/L235A/G236del/S267K, and the firstvariant Fc domain comprises pI substitutions Q295E/N384D/Q418D/N421D,wherein numbering is according to EU numbering.

In an exemplary embodiment of the “central-IL-15/Rα” formatheterodimeric protein, the first variant Fc domain comprises skewvariants S364K/E357Q and the second variant Fc domain comprise the skewvariant pair L368D/K370S, the first and second variant Fc domains eachcomprise FcKO variants E233P/L234V/L235A/G236del/S267K, and the secondvariant Fc domain of the second monomer comprises pI substitutionsQ295E/N384D/Q418D/N421D, wherein numbering is according to EU numbering.In some embodiments of the “central-IL-15/Rα” format heterodimericprotein, the hinge of the first and second monomers each comprise aminoacid substitution C220S, wherein numbering is according to EU numbering.In certain embodiments, the first and second variant Fc domains eachfurther comprise half-life extension variants M428:/N434S.

In another aspect, provided herein are “central-scIL-15/Rα” formatheterodimeric proteins. Such “central-scIL-15/Rα” format heterodimericproteins include: a) a first monomer comprising, from N-to C-terminal,VH-CH1-domain linker-IL-15Rα(sushi) domain-domain linker-IL-15variant-hinge-CH2-CH3, wherein the CH2-CH3 is a first variant Fc domain;b) a second monomer comprising, from N-to C-terminal, aVH-CH1-hinge-CH2-CH3, wherein the CH2-CH3 is a second variant Fc domain;and c) a third and fourth monomer that each comprises, from N-toC-terminal, VL-CL. The VH of the first monomer and the VL of the thirdmonomer form a first human LAG-3 binding domain, and the VH of thesecond monomer and the VL of the fourth monomer form a second humanLAG-3 binding domain.

In some embodiments of the “central-scIL-15/Rα” format heterodimericprotein, the first variant Fc domain the second variant Fc domaincomprises one of the following skew variant sets:S267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering. In an exemplary embodiment, the skew variant set isS364K/E357Q:L368D/K370S.

In an exemplary embodiment, the first variant Fc domain comprises skewvariants L368D/K370S and the second variant Fc domain comprises skewvariants S364K/E357Q, the first and second variant Fc domains eachcomprise FcKO variants E233P/L234V/L235A/G236del/S267K, and the firstvariant Fc domain comprises pI variants Q295E/N384D/Q418E/N421D, whereinnumbering is according to EU numbering. In some embodiments, the hingeof the first monomer comprises amino acid substitution C220S, whereinnumbering is according to EU numbering. In certain embodiments, thefirst and second variant Fc domains each further comprise half-lifeextension variants M428:/N434S.

In certain embodiment, the VH and VL of any of the heterodimericproteins provided herein are the variable heavy domain and variabledomain of any of the LAG-3 antigen binding domains in FIGS. 12 and 13.In an exemplary embodiment, the LAG-3 antigen binding domain is2A11_H1.144_L2.142 and 7G8_H3.30_L1.34.

In some embodiments, the IL-15 variant of the heterodimeric proteinprovided herein comprises an amino acid substitution(s) selected fromthe group consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, Q108E,N4D/N65D, D30N/N65D, and D30N/E64Q/N65D. In an exemplary embodiment, theIL-15 variant comprises amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D

In one aspect, provided herein is a pharmaceutical composition thatincludes any of the heterodimeric proteins disclosed herein and apharmaceutically acceptable carrier.

In another aspect, provided herein is a method of treating a patient inneed thereof comprising administering to the patient any one of theheterodimeric proteins or pharmaceutical compositions disclosed herein.In some embodiments, the method further comprising administering anantibody, where the antibody is an anti-PD-1 antibody, an anti-PD-L1antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody oran-anti-TIGIT antibody.

In another aspect, provided herein are nucleic acid compositions thatinclude one or more nucleic acids encoding any of the heterodimericproteins disclosed herein, expression vectors that include the nucleicacids, host cells that include the nucleic acids or expression vectors.Also provided herein are methods of making subject heterodimericproteins by culturing host cells under suitable conditions andrecovering the heterodimeric proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts selectivity of LAG-3-targeted IL-15/Rα-Fc fusion proteinsfor tumor-reactive tumor-infiltrating lymphocytes expressing PD-1, andits combination with PD-1 blockade antibody.

FIGS. 2A-2B depict the sequences for IL-15 and its receptors.

FIG. 3 depicts the sequences for LAG-3, including both human and cyno(predicted), to facilitate the development of antigen binding domainsthat bind to both for ease of clinical development.

FIGS. 4A-4E depict useful pairs of Fc heterodimerization variant sets(including skew and pI variants). There are variants for which there areno corresponding “monomer 2” variants; these are pI variants which canbe used alone on either monomer.

FIG. 5 depicts a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants of theinventions (and other variant types as well, as outlined herein.)

FIG. 6 depicts useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer.

FIGS. 7A-7F show particularly useful embodiments of“non-cytokine”/“non-Fv” components of the LAG-3-targeting IL-15/Rα-Fcfusion proteins of the invention.

FIG. 8 depicts a number of exemplary variable length linkers for use inIL-15/Rα-Fc fusion proteins. In some embodiments, these linkers find uselinking the C-terminus of IL-15 and/or IL-15Rα(sushi) to the N-terminusof the Fc region. In some embodiments, these linkers find use fusingIL-15 to the IL-15Rα(sushi).

FIGS. 9A-9C depict a number of charged scFv linkers that find use inincreasing or decreasing the pI of heterodimeric antibodies that utilizeone or more scFv as a component. The (+H) positive linker findsparticular use herein. A single prior art scFv linker with single chargeis referenced as “Whitlow”, from Whitlow et al., Protein Engineering6(8):989-995 (1993). It should be noted that this linker was used forreducing aggregation and enhancing proteolytic stability in scFvs.

FIG. 10 show the sequences of several useful LAG-3-targeting IL-15/Rα-Fcfusion format backbones based on human IgG1, without the cytokinesequences (e.g., the 11-15 and/or IL-15Rα(sushi)) or VH, and furtherexcluding light chain backbones which are depicted in FIG. 11. Backbone1 is based on human IgG1 (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, C220S and theQ295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370S skewvariants and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Backbone 2 is based on human IgG1 (356E/358M allotype), andincludes the S364K/E357Q:L368D/K370S skew variants, theN208D/Q295E/N384D/Q418E/N421D pI variants on the chain with L368D/K370Sskew variants, C220S in the chain with S364K/E357Q variants, and theE233P/L234V/L235A/G236del/S267K ablation variants on both chains.Backbone 3 is based on human IgG1 (356E/358M allotype), and includes theS364K/E357Q:L368D/K370S skew variants, the N208D/Q295E/N384D/Q418E/N421DpI variants on the chains with L368D/K370S skew variants, theQ196K/I199T/P217R/P228R/N276K pI variants on the chains with S364K/E357Qvariants, and the E233P/L234V/L235A/G236del/S267K ablation variants onboth chains. Such backbone sequences can be included, for example, inthe “scIL-15/Rα X Fab” format heterodimeric proteins described herein.).In some embodiments, the “scIL-15/Rα X Fab” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where hinge-CH2-CH3 has the amino acid sequence of “Chain 2” of any ofthe backbone sequences in FIG. 10 (SEQ ID NO: 58, 60 and 62); b) asecond monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain andCH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of any one ofthe backbone sequences in FIG. 10 (SEQ ID NO: 57, 59 and 61), and c) alight chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain and VC has the sequence of “Constant LightChain—Kappa” or “Constant Light Chain—Lambda “in FIG. 11 (SEQ ID NO:63-64). In an exemplary embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In exemplaryembodiments, the VH and VL are the variable heavy domain and variablelight domain, respectively, of any of the LAG-3 ABDs provided in FIGS.12 and 13A-C.

In certain embodiments, these sequences can be of the 356D/358Lallotype. In other embodiments, these sequences can include either theN297A or N297S substitutions. In some other embodiments, these sequencescan include the M428L/N434S Xtend mutations. In yet other embodiments,these sequences can instead be based on human IgG4, and include a S228P(EU numbering, this is S241P in Kabat) variant on both chains thatablates Fab arm exchange as is known in the art. In yet furtherembodiments, these sequences can instead be based on human IgG2.Further, these sequences may instead utilize the other skew variants, pIvariants, and ablation variants depicted in the Figures.

As will be appreciated by those in the art and outlined below, thesesequences can be used with any IL-15 and IL-15Rα(sushi) pairs outlinedherein, including but not limited to scIL-15/Rα, ncIL-15/Rα, anddsIL-15Rα, as schematically depicted in FIG. 21. Further as will beappreciated by those in the art and outlined below, any IL-15 and/orIL-15Rα(sushi) variants can be incorporated in these backbones.Furthermore as will be appreciated by those in the art and outlinedbelow, these sequences can be used with any VH and VL pairs outlinedherein, including either a scFv or a Fab.

Included within each of these backbones are sequences that are 90, 95,98 and 99% identical (as defined herein) to the recited sequences,and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions (as compared to the “parent” of the Figure, which, aswill be appreciated by those in the art, already contain a number ofamino acid modifications as compared to the parental human IgG1 (or IgG2or IgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition to the skew, pI and ablation variantscontained within the backbones of this figure.

FIG. 11 depicts the “non-Fv” backbone of light chains (i.e. constantlight chain) which find use in LAG-3-targeting IL-15/Rα-Fc fusionproteins of the invention.

FIG. 12 depicts the variable region sequences for a select number ofanti-LAG-3 antibody binding domains. The CDRs are underlined. As notedherein and is true for every sequence herein containing CDRs, the exactidentification of the CDR locations may be slightly different dependingon the numbering used as is shown in Table 2, and thus included hereinare not only the CDRs that are underlined but also CDRs included withinthe VH and VL domains using other numbering systems. Furthermore, as forall the sequences in the Figures, these VH and VL sequences can be usedeither in a scFv format or in a Fab format.

FIGS. 13A-13C depict the variable regions of additional LAG-3 ABDs whichmay find use in the LAG-3-targeting IL-15/Rα-Fc fusion proteins of theinvention. The CDRs are underlined. As noted herein and is true forevery sequence herein containing CDRs, the exact identification of theCDR locations may be slightly different depending on the numbering usedas is shown in Table 2, and thus included herein are not only the CDRsthat are underlined but also CDRs included within the VH and VL domainsusing other numbering systems. Furthermore, as for all the sequences inthe Figures, these VH and VL sequences can be used either in a scFvformat or in a Fab format.

FIG. 14 depicts a structural model of the IL-15/Rα heterodimer showinglocations of engineered disulfide bond pairs.

FIG. 15 depicts sequences for illustrative IL-15Rα(sushi) variantsengineered with additional residues at the C-terminus to serve as ascaffold for engineering cysteine residues.

FIG. 16 depicts sequences for illustrative IL-15 variants engineeredwith cysteines in order to form covalent disulfide bonds withIL-15Rα(sushi) variants engineered with cysteines.

FIG. 17 depicts sequences for illustrative IL-15Rα(sushi) variantsengineered with cysteines in order to form covalent disulfide bonds withIL-15 variants engineered with cysteines.

FIG. 18 depicts the structure of IL-15 complexed with IL-15Rα, IL-2Rβ,and common gamma chain. Locations of substitutions designed to reducepotency are shown.

FIGS. 19A-19C depict sequences for illustrative IL-15 variantsengineered for reduced potency. Included within each of these variantIL-15 sequences are sequences that are 90, 95, 98 and 99% identical (asdefined herein) to the recited sequences, and/or contain from 1, 2, 3,4, 5, 6, 7, 8, 9 or 10 additional amino acid substitutions. In anon-limiting example, the recited sequences may contain additional aminoacid modifications such as those contributing to formation of covalentdisulfide bonds as shown in FIG. 14, FIG. 16, and FIG. 17.

FIG. 20 depicts EC50 for induction of NK and CD8⁺ T cells proliferationby variant IL-15/Rα-Fc fusion proteins, and fold reduction in EC50relative to XENP20818, the wild type. These fusion proteins do notcontain a LAG-3 ABD.

FIG. 21A-FIG. 21K depict several formats for the LAG-3-targetingIL-15/Rα-Fc fusion proteins of the present invention. The “scIL-15/Rα xscFv” format (FIG. 21A) comprises IL-15Rα(sushi) fused to IL-15 by avariable length linker (termed “scIL-15/Rα”) which is then fused to theN-terminus of a heterodimeric Fc-region, with an scFv fused to the otherside of the heterodimeric Fc. The “scFv x ncIL-15/Rα” format (FIG. 21B)comprises an scFv fused to the N-terminus of a heterodimeric Fc-region,with IL-15Rα(sushi) fused to the other side of the heterodimeric Fc,while IL-15 is transfected separately so that a non-covalent IL-15/Rαcomplex is formed. The “scFv x dsIL-15/Rα” format (FIG. 21C) is the sameas the “scFv x ncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15are covalently linked as a result of engineered cysteines. The“scIL-15/Rα x Fab” format (FIG. 21D) comprises IL-15Rα(sushi) fused toIL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with a variableheavy chain (VH) fused to the other side of the heterodimeric Fc, whilea corresponding light chain is transfected separately so as to form aFab with the VH. The “ncIL-15/Rα x Fab” format (FIG. 21E) comprises a VHfused to the N-terminus of a heterodimeric Fc-region, withIL-15Rα(sushi) fused to the other side of the heterodimeric Fc, while acorresponding light chain is transfected separately so as to form a Fabwith the VH, and while IL-15 is transfected separately so that anon-covalent IL-15/Rα complex is formed. The “dsIL-15/Rα x Fab” format(FIG. 21F) is the same as the “ncIL-15/Rα x Fab” format, but whereinIL-15Rα(sushi) and IL-15 are covalently linked as a result of engineeredcysteines. The “mAb-scIL-15/Rα” format (FIG. 21G) comprises VH fused tothe N-terminus of a first and a second heterodimeric Fc, with IL-15 isfused to IL-15Rα(sushi) which is then further fused to the C-terminus ofone of the heterodimeric Fc-region, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs. The“mAb-ncIL-15/Rα” format (FIG. 21H) comprises VH fused to the N-terminusof a first and a second heterodimeric Fc, with IL-15Rα(sushi) fused tothe C-terminus of one of the heterodimeric Fc-region, whilecorresponding light chains are transfected separately so as to form aFabs with the VHs, and while and while IL-15 is transfected separatelyso that a non-covalent IL-15/Rα complex is formed. The “mAb-dsIL-15/Rα”format (FIG. 21I) is the same as the “mAb-ncIL-15/Rα” format, butwherein IL-15Rα(sushi) and IL-15 are covalently linked as a result ofengineered cysteines. The “central-IL-15/Rα” format (FIG. 21J) comprisesa VH recombinantly fused to the N-terminus of IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH recombinantlyfused to the N-terminus of IL-15Rα(sushi) which is then further fused tothe other side of the heterodimeric Fc, while corresponding light chainsare transfected separately so as to form a Fabs with the VHs. The“central-scIL-15/Rα” format (FIG. 21K) comprises a VH fused to theN-terminus of IL-15Rα(sushi) which is fused to IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH fused to theother side of the heterodimeric Fc, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs.

FIGS. 22A-22B depict sequences of XENP27972 and XENP27973, illustrativeLAG-3-targeting IL-15/Rα-Fc fusion protein of the “scIL-15/Rα x Fab”format. The CDRs are in bold. As noted herein and is true for everysequence herein containing CDRs, the exact identification of the CDRlocations may be slightly different depending on the numbering used asis shown in Table 2, and thus included herein are not only the CDRs thatare underlined but also CDRs included within the V_(H) and V_(L) domainsusing other numbering systems. IL-15 and IL-15Rα(sushi) are underlined,linkers are double underlined (although as will be appreciated by thosein the art, the linkers can be replaced by other linkers, some of whichare depicted in the Figures, and slashes (/) indicate the border(s)between IL-15, IL-15Rα, linkers, variable regions, and constant/Fcregions.

FIG. 23 depict the sequences for XENP16432, a bivalent anti-PD-1 mAbwith an ablation variant (E233P/L234V/L235A/G236del/S267K,“IgG1_PVA_/S267k”). The CDRs are underlined. As noted herein and is truefor every sequence herein containing CDRs, the exact identification ofthe CDR locations may be slightly different depending on the numberingused as is shown in Table 2, and thus included herein are not only theCDRs that are underlined but also CDRs included within the VH and VLdomains using other numbering systems.

FIGS. 24A-24B depict CD8⁺ T cell counts in whole blood of PBMC-engraftedNSG mice on Days A) 6 and B) 10 after first dose of the indicated testarticles.

FIGS. 25A-25B depict CD4⁺ T cell counts in whole blood of PBMC-engraftedNSG mice on Days A) 6 and B) 10 after first dose of the indicated testarticles.

FIGS. 26A-26B depict CD45⁺ T cell counts in whole blood ofPBMC-engrafted NSG mice on Days A) 6 and B) 10 after first dose of theindicated test articles.

FIGS. 27A-27B depict CD16⁺CD56⁺ NK cell counts in whole blood ofPBMC-engrafted NSG mice on Days A) 6 and B) 10 after first dose of theindicated test articles.

FIG. 28 depicts the change in body weight (as percentage of initial bodyweight) of PBMC-engrafted NSG mice after dosing with the indicated testarticles.

FIGS. 29A-29B depict the sequences of XENP27977 and 27978 that includeM428L/N434S variants in both Fc domains.

FIG. 30 depicts induction of A) CD8⁺ T cells and B) CD4⁺ T cellsproliferation by LAG-3-targeted IL-15/Rα-Fc fusions (and controls) asindicated by percentage proliferating cells (determined based on CFSEdilution). The data show that LAG-3-targeted IL-15/Rα-Fc fusions aremore potent in inducing proliferation of CD8⁺ T cells in comparison tountargeted IL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as well as controlRSV-targeted IL-15/Rα-Fc fusion).

FIG. 31 depicts induction of A) CD8 memory T cell and B) CD8 naive Tcell proliferation by LAG-3-targeted IL-15/Rα-Fc fusions (and controls)as indicated by percentage proliferating cells (determined based on CFSEdilution). The data show that LAG-3-targeted IL-15/Rα-Fc fusions aremuch more potent in inducing proliferation of CD8 memory T cells incomparison to untargeted IL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as well ascontrol RSV-targeted IL-15/Rα-Fc fusion).

FIG. 32 depicts induction of A) CD8 memory T cell and B) CD8 naive Tcell proliferation by LAG-3-targeted IL-15/Rα-Fc fusions (and controls)as indicated by cell counts. The data show that LAG-3-targetedIL-15/Rα-Fc fusions are much more potent in expanding CD8 memory T cellsin comparison to untargeted IL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as wellas control RSV-targeted IL-15/Rα-Fc fusion).

FIG. 33 depicts induction of A) CD4 memory T cell and B) CD4 naive Tcell proliferation by LAG-3-targeted IL-15/Rα-Fc fusions (and controls)as indicated by percentage proliferating cells (determined based on CFSEdilution). The data show that LAG-3-targeted IL-15/Rα-Fc fusions aremore potent in expanding CD4 memory T cells in comparison to untargetedIL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as well as control RSV-targetedIL-15/Rα-Fc fusion).

FIG. 34 depicts induction of A) CD4 memory T cell and B) CD4 naive Tcell proliferation by LAG-3-targeted IL-15/Rα-Fc fusions (and controls)as indicated by cell counts.

FIG. 35 depicts induction of NK cells proliferation by LAG-3-targetedIL-15/Rα-Fc fusions (and controls) as indicated A) percentageproliferating cells (determined based on CFSE dilution) and B) by cellcounts.

FIG. 36 depicts activation of CD8⁺ T cells as indicated by A) percentageCD8 memory T cells expressing CD25, B) percentage CD8 naive T cellsexpressing CD25, C) percentage CD4 memory T cells expressing CD25, andD) percentage CD4 naive T cells expressing CD25 following incubationwith LAG-3-targeted IL-15/Rα-Fc fusions (and controls). The data showthat LAG-3-targeted IL-15/Rα-Fc fusions, in particular XENP27972, appearto upregulate CD25 in both CD8 and CD4 memory T cells in comparison tountargeted IL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as well as controlRSV-targeted IL-15/Rα-Fc fusion).

FIG. 37 depicts activation of CD8⁺ T cells as indicated by A) HLA-DR MFIon CD8 memory T cells, B) percentage CD8 memory T cells expressingHLA-DR, C) HLA-DR MFI on CD8 naive T cells, and D) percentage CD8 naiveT cells expressing HLA-DR following incubation with LAG-3-targetedIL-15/Rα-Fc fusions (and controls). The data show that LAG-3-targetedIL-15/Rα-Fc fusions appear to upregulate HLA-DR in CD8 memory T cellsmore potently in comparison to untargeted IL-15(D30N/E64Q/N65D)/Rα-Fcfusion (as well as control RSV-targeted IL-15/Rα-Fc fusion).

FIG. 38 depicts activation of CD4⁺ T cells as indicated by A) HLA-DR MFIon CD4 memory T cells, B) percentage CD4 memory T cells expressingHLA-DR, C) HLA-DR MFI on CD4 naive T cells, and D) percentage CD4 naiveT cells expressing HLA-DR following incubation with LAG-3-targetedIL-15/Rα-Fc fusions (and controls).

FIG. 39 depicts the sequences of XENP22853, an IL-15/Rα-heteroFc fusioncomprising a wild-type IL-15 and Xtend Fc (M428L/N434S) variant. IL-15and IL-15Rα(sushi) are underlined, linkers are double underlined(although as will be appreciated by those in the art, the linkers can bereplaced by other linkers, some of which are depicted in the Figures,and slashes (/) indicate the border(s) between IL-15, IL-15Rα, linkers,and constant/Fc regions.

FIG. 40 depicts the sequences of XENP24113, an IL-15/Rα-heteroFc fusioncomprising an IL-15(N4D/N65D) variant and Xtend Fc (M428L/N434S)variant. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inthe Figures, and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, and constant/Fc regions.

FIG. 41 depicts the sequences of XENP24294, an scIL-15/Rα-Fc fusioncomprising an IL-15(N4D/N65D) variant and Xtend Fc (M428L/N434S)substitution. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin the Figures, and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, and constant/Fc regions.

FIG. 42 depicts the sequences of XENP24306, an IL-15/Rα-heteroFc fusioncomprising an IL-15(D30N/E64Q/N65D) variant and Xtend Fc (M428L/N434S)substitution. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin the Figures, and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, and constant/Fc regions.

FIG. 43 depicts the serum concentration of the indicated test articlesover time in cynomolgus monkeys following a first dose at the indicatedrelative concentrations.

FIGS. 44A-44C depict sequences of illustrative scIL-15/Rα-Fc fusionscomprising additional IL-15 potency variants. IL-15 and IL-15Rα(sushi)are underlined, linkers are double underlined (although as will beappreciated by those in the art, the linkers can be replaced by otherlinkers, some of which are depicted in Figures some of which aredepicted in FIGS. 9 and 10), and slashes (/) indicate the border(s)between IL-15, IL-15Rα, linkers, variable regions, and constant/Fcregions.

FIGS. 45A-45G depicts percentage of A) CD4⁺CD45RA⁻, B) CD4⁺CD45RA⁺, C)CD8⁺CD45RA⁻, D) CD8⁺CD45RA⁺, E) CD16⁺ NK cells, F) CD56⁺ NK cells, andG) γδ cells expression Ki67 following incubation of PBMCs with theindicated test articles for 3 days.

FIGS. 46A and 46B depict sequences of illustrative LAG-3-targetedIL-15/Rα-Fc fusions comprising IL-15(D30N/N65D) variant. The CDRs are inbold. As noted herein and is true for every sequence herein containingCDRs, the exact identification of the CDR locations may be slightlydifferent depending on the numbering used as is shown in Table 2, andthus included herein are not only the CDRs that are underlined but alsoCDRs included within the V_(H) and V_(L) domains using other numberingsystems. IL-15 and IL-15Rα(sushi) are underlined, linkers are doubleunderlined (although as will be appreciated by those in the art, thelinkers can be replaced by other linkers, some of which are depicted inFIGS. 9 and 10), and slashes (/) indicate the border(s) between IL-15,IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIGS. 47A and 47B depicts sequences of illustrative LAG-3-targetedIL-15/Rα-Fc fusions comprising IL-15(D30N/E64Q/N65D) variant. The CDRsare in bold. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table2, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the VH and VL domains using othernumbering systems. IL-15 and IL-15Rα(sushi) are underlined, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin FIGS. 9 and 10), and slashes (/) indicate the border(s) betweenIL-15, IL-15Rα, linkers, variable regions, and constant/Fc regions.

FIGS. 48A-48D depict sequences of illustrative LAG-3-targetedIL-15/Rα-Fc fusions comprising Xtend (M428L/N434S) substitutions forenhancing serum half-life. The CDRs are in bold. As noted herein and istrue for every sequence herein containing CDRs, the exact identificationof the CDR locations may be slightly different depending on thenumbering used as is shown in Table 2, and thus included herein are notonly the CDRs that are underlined but also CDRs included within theV_(H) and V_(L) domains using other numbering systems. IL-15 andIL-15Rα(sushi) are underlined, linkers are double underlined (althoughas will be appreciated by those in the art, the linkers can be replacedby other linkers, some of which are depicted in FIGS. 9 and 10), andslashes (/) indicate the border(s) between IL-15, IL-15Rα, linkers,variable regions, and constant/Fc regions. It should be noted that anyof the sequences depicted herein may include or exclude the M428L/N434Ssubstitutions.

FIGS. 49A and 49B depict the sequences of XENP26007, XENP29481, andXENP30432, control RSV-targeted IL-15/Rα-Fc fusions. The CDRs areunderlined. As noted herein and is true for every sequence hereincontaining CDRs, the exact identification of the CDR locations may beslightly different depending on the numbering used as is shown in Table2, and thus included herein are not only the CDRs that are underlinedbut also CDRs included within the V_(H) and V_(L) domains using othernumbering systems. IL-15 and IL-15Rα(sushi) are italicized, linkers aredouble underlined (although as will be appreciated by those in the art,the linkers can be replaced by other linkers, some of which are depictedin Figures some of which are depicted in FIGS. 9 and 10), and slashes(/) indicate the border(s) between IL-15, IL-15Rα, linkers, variableregions, and constant/Fc regions.

DETAILED DESCRIPTION I. DEFINITIONS

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding are those shown in FIG. 6. However,unless otherwise noted, the Fc monomers of the invention retain bindingto the FcRn receptor.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity. As is discussed herein, many embodiments of theinvention ablate ADCC activity entirely.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, a “LAG-3 antigen binding domain” binds a human LAG-3antigen as outlined herein. As is known in the art, these CDRs aregenerally present as a first set of variable heavy CDRs (vhCDRs orV_(H)CDRs) and a second set of variable light CDRs (vlCDRs orV_(L)CDRs), each comprising three CDRs: vhCDR1, vhCDR2, vhCDR3 for theheavy chain and vlCDR1, vlCDR2 and vlCDR3 for the light. The CDRs arepresent in the variable heavy and variable light domains, respectively,and together form an Fv region. Thus, in some cases, the six CDRs of theantigen binding domain are contributed by a variable heavy and variablelight chain. In a “Fab” format, the set of 6 CDRs are contributed by twodifferent polypeptide sequences, the variable heavy domain (VH or vh orV_(H); containing the vhCDR1, vhCDR2 and vhCDR3) and the variable lightdomain (VL or vl or V_(L); containing the vlCDR1, vlCDR2 and vlCDR3),with the C-terminus of the VH domain being attached to the N-terminus ofthe CH1 domain of the heavy chain and the C-terminus of the vl domainbeing attached to the N-terminus of the constant light domain (and thusforming the light chain). In a scFv format, the VH and VL domains arecovalently attached, generally through the use of a linker as outlinedherein, into a single polypeptide sequence, which can be either(starting from the N-terminus) VH-linker-VL or VL-linker-VH, with theformer being generally preferred (including optional domain linkers oneach side, depending on the format used (e.g., from FIG. 1 of U.S.62/353,511).

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g., the 20 amino acids that have codonsin DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233− or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233− or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g., fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4. The protein variantsequence herein will preferably possess at least about 80% identity witha parent protein sequence, and most preferably at least about 90%identity, more preferably at least about 95-98-99% identity. Variantprotein can refer to the variant protein itself, compositions comprisingthe protein variant, or the DNA sequence that encodes it.

“Variant,” as used herein can also refer to particular amino acidmodifications (e.g., substitutions, deletions, insertions) in a variantprotein (e.g., a variant Fc domain), for example, heterodimerizationvariants, ablation variants, FcKO variants, etc., as disclosed inSection II.C. below.

Accordingly, by “Fc variant” or “variant Fc” as used herein is meant aprotein comprising an amino acid modification in an Fc domain. The Fcvariants of the present invention are defined according to the aminoacid modifications that compose them. Thus, for example, N434S or 434Sis an Fc variant with the substitution serine at position 434 relativeto the parent Fc polypeptide, wherein the numbering is according to theEU index. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S relative to the parent Fc polypeptide. Theidentity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention that relateto antibodies, unless otherwise noted, amino acid position numbering isaccording to the EU index. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference). The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand, in some cases, synthetic amino acids. Examples include U.S. Pat.No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of theAmerican Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz,(2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICASUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz,(2002), Chem. 1-10, all entirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of afull-length antibody, antibody fragment or Fab fusion protein.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.As will be appreciated by those in the art, these generally are made upof two chains, or can be combined (generally with a linker as discussedherein) to form an scFv.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (VH-linker-VL orVL-linker-VH).

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. As is known in the art, the functional FcRn proteincomprises two polypeptides, often referred to as the heavy chain andlight chain. The light chain is beta-2-microglobulin and the heavy chainis encoded by the FcRn gene. Unless otherwise noted herein, FcRn or anFcRn protein refers to the complex of FcRn heavy chain withbeta-2-microglobulin. A variety of FcRn variants can be used to increasebinding to the FcRn receptor, and in some cases, to increase serumhalf-life. In general, unless otherwise noted, the Fc monomers of theinvention retain binding to the FcRn receptor (and, as noted below, caninclude amino acid variants to increase binding to the FcRn receptor).

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain (e.g., CH1) and in somecases, part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hingeregion between CH1 (Cγ1) and CH2 (Cγ2). Thus, in some cases, the Fcdomain includes, from N- to C-terminal, CH2-CH3 and hinge-CH2-CH3. Insome embodiments, the Fc domain is that from IgG1, IgG2, IgG3 or IgG4,with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3 finding particular use inmany embodiments. Additionally, in certain embodiments, wherein the Fcdomain is a human IgG1 Fc domain, the hinge includes a C220S amino acidsubstitution. Furthermore, in some emboidments where the Fc domain is ahuman IgG4 Fc domain, the hinge includes a S228P amino acidsubstitution. Although the boundaries of the Fc region may vary, thehuman IgG heavy chain Fc region is usually defined to include residuesC226 or P230 to its carboxyl-terminus, wherein the numbering isaccording to the EU index as in Kabat. Accordingly, “CH” domains in thecontext of IgG are as follows: “CH1” refers to positions 118-215according to the EU index as in Kabat. “Hinge” refers to positions216-230 according to the EU index as in Kabat. “CH2” refers to positions231-340 according to the EU index as in Kabat, and “CH3” refers topositions 341-447 according to the EU index as in Kabat. Thus, the “Fcdomain” includes the -CH2-CH3 domain, and optionally a hinge domain(hinge-CH2-CH3).

As will be appreciated by those in the art, the exact numbering andplacement of the heavy constant region domains can be different amongdifferent numbering systems. A useful comparison of heavy constantregion numbering according to EU and Kabat is as below, see Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, entirelyincorporated by reference.

TABLE 1 EU Numbering Kabat Numbering CH1 118-215 114-223 Hinge 216-230226-243 CH2 231-340 244-360 CH3 341-447 361-478

In the embodiments herein, when a scFv or IL-15 complex is attached toan Fc domain, it is the C-terminus of the scFv, IL-15 or IL-15Rαconstruct that is attached to the Fc domain via a domain linker; forexample, a hinge domain as depicted in FIG. 8. In some embodiments, asis more fully described below, amino acid modifications are made to theFc region, for example to alter binding to one or more FcγR receptors orto the FcRn receptor, and to enable heterodimer formation andpurification, as outlined herein.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof an antibody.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as to IL-15and/or IL-15R, as described herein. In some instances, two Fc fusionproteins can form a homodimeric Fc fusion protein or a heterodimericfusion protein with the latter being preferred. In some cases, onemonomer of the heterodimeric fusion protein comprises an Fc domain alone(e.g., an empty Fc domain) and the other monomer is a Fc fusion,comprising a variant Fc domain and a protein domain, such as a receptor,ligand or other binding partner.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “strandedness” in the context of the monomers of the heterodimericantibodies of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g., making the pI higher) then steric variants thatare “charge pairs” that can be utilized as well do not interfere withthe pI variants, e.g., the charge variants that make a pI higher are puton the same “strand” or “monomer” to preserve both functionalities.Similarly, for “skew” variants that come in pairs of a set as more fullyoutlined below, the skilled artisan will consider pI in deciding intowhich strand or monomer that incorporates one set of the pair will go,such that pI separation is maximized using the pI of the skews as well.

By “target cell” as used herein is meant a cell that expresses thetarget antigen, in this case, LAG-3.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The subject LAG-3 targeted heterodimeric proteins are generally isolatedor recombinant. “Isolated,” when used to describe the variouspolypeptides disclosed herein, means a polypeptide that has beenidentified and separated and/or recovered from a cell or cell culturefrom which it was expressed. Ordinarily, an isolated polypeptide will beprepared by at least one purification step. An “isolated protein,”refers to a protein which is substantially free of other proteins havingdifferent binding specificities. “Recombinant” means the proteins aregenerated using recombinant nucleic acid techniques in exogeneous hostcells.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Pub. No. 20160244525, hereby incorporated byreference.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope (in this case, human LAG-3) meansbinding that is measurably different from a non-specific interaction.Specific binding can be measured, for example, by determining binding ofa molecule compared to binding of a control molecule, which generally isa molecule of similar structure that does not have binding activity. Forexample, specific binding can be determined by competition with acontrol molecule that is similar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸M, at least about 10⁻⁹M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹³M, at leastabout 10⁻¹²M, or greater, where KD refers to a dissociation rate of aparticular antibody-antigen interaction. Typically, an antibody thatspecifically binds an antigen will have a KD that is 20-, 50-, 100-,500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction. Binding affinity is generally measured using a Biacoreassay.

II. INTRODUCTION

The invention provides heterodimeric fusion proteins that contain anIL-15 complex on one side and an anti-human LAG-3 antigen binding domainon the other. Thus, the heterodimeric fusion proteins of the inventioncan bind to the checkpoint LAG-3 antigen and can complex with the commongamma chain (γc; CD132) and/or the IL-2 receptor β-chain (IL-2Rβ;CD122). In general, the heterodimeric fusion proteins of the inventionhave three functional components: an IL-15/IL-15Rα(sushi) component,generally referred to herein as an “IL-15 complex”, an anti-LAG-3 ABDcomponent which serves as a “targeting” moiety by bringing the fusionprotein to a cell expressing LAG-3, and an Fc component, each of whichcan take different forms and each of which can be combined with theother components in any configuration.

In general, as is more fully described herein, the fusion proteins ofthe invention are heterodimeric proteins that are based on theassociation of antibody Fc domains. That is, by using two differentvariant Fc domains that have been engineered to favor the formation ofheterodimers over homodimers, the heterodimeric proteins are formed. Inthis case, one of the variant Fc domains is fused to an IL-15/RA complexand the other has a LAG-3 ABD as more fully outlined herein. Byincluding optional pI variants, the heterodimers can be more easilypurified away from the homodimers. Additionally, the inclusion ofablation variants eliminates the effector functions of the Fc domains.

A. IL-15/IL-15Rα(Sushi) Domains

As shown in the figures, the IL-15 complex can take several forms. Asstated above, the IL-15 protein on its own is less stable than whencomplexed with the IL-15Rα protein. As is known in the art, the IL-15Rαprotein contains a “sushi domain”, which is the shortest region of thereceptor that retains IL-15 binding activity. Thus, while heterodimericfusion proteins comprising the entire IL-15Rα protein can be made,preferred embodiments herein include complexes that just use the sushidomain, the sequence of which is shown in the figures.

Accordingly, the IL-15 complex generally comprises the IL-15 protein andthe sushi domain of IL IL-15Rα (unless otherwise noted that the fulllength sequence is used, “IL-15Rα”, “IL-15Rα(sushi)”, “IL-15RA” and“sushi” are used interchangeably throughout).

Importantly, the IL-15 component is generally engineered to reduce itspotency. In many embodiments, the wild-type IL-15 is too potent and cancause undesirable toxicity. Accordingly, the IL-15 component of theIL-15 complex can have one or more amino acid substitutions that resultin decreased activity. Various amino acid substitutions were made (seeFIG. 19) and tested (see FIG. 20). Of particular interest in someembodiments are a double variant, N4D/N65D or D30N/N65D, or a triplevariant, D30N/E64Q/N65D.

The targeted IL-15/IL-15Rα heterodimeric fusion proteins of the presentinvention include an IL-15/IL-15 receptor alpha (IL-15Rα)-Fc fusionmonomer; reference is made to US2018/0118828, filed 16, October 2017,U.S. Ser. No. 62/408,655, filed on Oct. 14, 2016, U.S. Ser. No.62/416,087, filed on October Nov. 1, 2016, U.S. Ser. No. 62/443,465,filed on Jan. 6, 2017, U.S. Ser. No. 62/477,926, filed on Mar. 28, 2017,and U.S. Ser. No. 62/659,571, filed on Apr. 18, 2018, herebyincorporated by reference in their entirety and in particular for thesequences outlined therein. In some cases, the IL-15 and IL-15 receptoralpha (IL-15Rα) protein domains are in different orientations. Exemplaryembodiments of IL-15/IL-15Rα-Fc fusion monomers are provided inXENP21480 (chain 1; FIG. 64A), XENP22022 (chain 1, FIG. 64D), XENP22112,(chains 1 and 3; FIG. 64E), XENP22641 (chains 2 and 4; FIG. 64F),XENP22642, (chains 1 and 4; FIG. 64H) and XENP22644 (chains 1 and 4;FIG. 64I) as described, for example, in US 2018/0118828.

1. IL-15 Variants

In some embodiments, the human IL-15 protein has the amino acid sequenceset forth in NCBI Ref. Seq. No. NP_000576.1 as shown in FIG. 2. In somecases, the coding sequence of human IL-15 is set forth in NCBI Ref. Seq.No. NM_000585. An exemplary IL-15 protein of the Fc fusion heterodimericprotein outlined herein can have the amino acid sequence of SEQ ID NO:2or amino acids 49-162 of SEQ ID NO:1. In some embodiments, the IL-15protein has at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more sequence identity to SEQ ID NO:2. In some embodiments,the IL-15 protein has the amino acid sequence set forth in SEQ ID NO:2except with the amino acid substitution N72D. In other embodiments, theIL-15 protein has the amino acid sequence of SEQ ID NO:2 except with oneor more amino acid substitutions selected from the group consisting ofC42S, L45C, Q48C, V49C, L52C, E53C, E87C, and E89C. In some aspects, theIL-15 protein has one or more amino acid substitutions selected from thegroup consisting of N1D, N4D, D8N, D30N, D61N, E64Q, N65D, and Q108E. Inother embodiments, the amino acid substitutions are N4D/N65D. In certainembodiments, the amino acid substitutions are D30N/N65D. In someembodiments, the amino acid substitution is Q108E. In certainembodiments, the amino acid substitution is N65D. In other embodiments,the amino acid substitutions are D30N/E64Q/N65D. In certain embodiments,the amino acid substitution is N65D. In some instances, the amino acidsubstitutions are N1D/N65D. In some instances, the amino acidsubstitutions are D30N/N65D. Optionally, the IL-15 protein also has anN72D substitution. The IL-15 protein of the Fc fusion protein can have1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions. In someembodiments, the IL-15 protein of the Fc fusion protein comprises a D30Nsubstitution. In some embodiments, the IL-15 protein of the Fc fusionprotein comprises a N65D substitution. In some embodiments, the IL-15protein of the Fc fusion contains one or more amino acid substitutionsat the IL-15:CD132 interface. In certain embodiments, the Fc fusionprotein described herein induces proliferation of NK cells and CD8+ Tcells. Additionally, IL-15 variants that can be used with the subjecttargeted IL-15/IL-15Rα heterodimer proteins are included in FIGS. 19A-C.

In some embodiments, the human IL-15 receptor alpha (IL-15Rα) proteinhas the amino acid sequence set forth in NCBI Ref. Seq. No. NP_002180.1or SEQ ID NO:3. In some cases, the coding sequence of human IL-15Rα isset forth in NCBI Ref. Seq. No. NM_002189.3. An exemplary the IL-15Rαprotein of the Fc fusion heterodimeric protein outlined herein cancomprise or consist of the sushi domain of SEQ ID NO:3 (e.g., aminoacids 31-95 of SEQ ID NO:3), or in other words, the amino acid sequenceof SEQ ID NO:4. In some embodiments, the IL-15Rα protein has the aminoacid sequence of SEQ ID NO:4 and an amino acid insertion selected fromthe group consisting of D96, P97, A98, D96/P97, D96/C97, D96/P97/A98,D96/P97/C98, and D96/C97/A98, wherein the amino acid position isrelative to full-length human IL-15Rα protein or SEQ ID NO:3. Forinstance, amino acid(s) such as D (e.g., Asp), P (e.g., Pro), A (e.g.,Ala), DP (e.g., Asp-Pro), DC (e.g., Asp-Cys), DPA (e.g., Asp-Pro-Ala),DPC (e.g., Asp-Pro-Cys), or DCA (e.g., Asp-Cys-Ala) can be added to theC-terminus of the IL-15Rα protein of SEQ ID NO:4. In some embodiments,the IL-15Rα protein has the amino acid sequence of SEQ ID NO:4 and oneor more amino acid substitutions selected from the group consisting ofK34C, A37C, G38C, S40C, and L42C, wherein the amino acid position isrelative to SEQ ID NO:4. The IL-15Rα protein can have 1, 2, 3, 4, 5, 6,7, 8 or more amino acid mutations (e.g., substitutions, insertionsand/or deletions).

2. IL-15/RA Complexes

As outlined herein, the IL-15 variants and the sushi domain can becomplexed in at least three different ways to form an IL-15 complex.

In some embodiments, as shown in FIG. 21B, for example, the IL-15protein and the IL-15Rα(sushi) are not covalently attached, but ratherare self-assembled through regular ligand-ligand interactions. As ismore fully described herein, it can be either the IL-15 domain or thesushi domain that is covalently linked to the Fc domain (generally usingan optional domain linker). Again, of particular use in this embodimentare a double variant, N4D/N65D or D30N/N65D, or a triple variant,D30N/E64Q/N65D, used with a wild type sushi domain.

In alternative embodiments, the variant IL-15 can be complexed to thesushi domain using a domain linker, such that they are covalentlyattached as generally shown in FIG. 21D; this figure depicts the sushidomain as the N-terminal domain, although this can be reversed. Again,of particular use in this embodiment are a double variant, N4D/N65D orD30N/N65D, or a triple variant, D30N/E64Q/N65D, used with a wild typesushi domain. Exemplary domain linkers that can be used to attach theIL-15 variant to the sushi domain are depicted in FIG. 8.

Alternatively, each of the IL-15 and sushi domains can be engineered tocontain a cysteine amino acid, that forms a disulfide bond to form thecomplex as is generally shown in FIG. 21C, again, with either the IL-15domain or the sushi domain being covalently attached (using an optionaldomain linker) to the Fc domain. Again, of particular use in thisembodiment are a double variant, N4D/N65D or D30N/N65D, (additionallyincluding an amino acid substitution to cysteine), or a triple variant,D30N/E64Q/N65D (additionally including an amino acid substitution tocysteine), used with a sushi domain also comprising an amino acidsubstitution to provide a cysteine.

Additional particular embodiments are outlined below.

B. Anti-LAG-3 Components

In some embodiments, the heterodimeric fusion proteins provided hereininclude some antibody components.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to antibodies or antibody fragments (antibody monomers) thatgenerally are based on the IgG class, which has several subclasses,including, but not limited to IgG1, IgG2, IgG3, and IgG4. In general,IgG1, IgG2 and IgG4 are used more frequently than IgG3. It should benoted that IgG1 has different allotypes with polymorphisms at 356 (D orE) and 358 (L or M). The sequences depicted herein use the 356D/358Mallotype, however the other allotype is included herein. That is, anysequence inclusive of an IgG1 Fc domain included herein can have356E/358L replacing the 356D/358M allotype.

In addition, many of the monomer sequences herein have at least one thecysteines at position 220 replaced by a serine, to reduce disulfideformation. Specifically included within the sequences herein are one orboth of these cysteines replaced (C220S).

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g., residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g., vhCDR1,vhCDR2 and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g., vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol. 27(1):55-77 (2003):

TABLE 2 Kabat + Chothia IMGT Kabat AbM Chothia Contact Xencor vhCDR126-35 27-38 31-35 26-35 26-32 30-35 27-35 vhCDR2 50-65 56-65 50-65 50-5852-56 47-58 54-61 vhCDR3  95-102 105-117  95-102  95-102  95-102  93-101103-116 vlCDR1 24-34 27-38 24-34 24-34 24-34 30-36 27-38 vlCDR2 50-5656-65 50-56 50-56 50-56 46-55 56-62 vlCDR3 89-97 105-117 89-97 89-9789-97 89-96  97-105

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g., Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g., a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.” As outlined below,the invention not only includes the enumerated antigen binding domainsand antibodies herein, but those that compete for binding with theepitopes bound by the enumerated antigen binding domains.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ).

Another region of interest for additional substitutions, outlinedherein, is the Fc region.

Thus, the present heterodimeric fusion proteins provided herein includeone or more antibody domains. As described herein and known in the art,the heterodimeric antibodies provided herein comprise different domainswithin the heavy and light chains, which can be overlapping as well.These domains include, but are not limited to, the Fc domain, the CH1domain, the CH2 domain, the CH3 domain, the hinge domain, the heavyconstant domain (CH1-hinge-Fc domain or CH1-hinge-CH2-CH3), the variableheavy domain, the variable light domain, the light constant domain, Fabdomains and scFv domains.

As generally outlined herein, the heterodimeric proteins of theinvention include one or more LAG-3 antigen binding domains (e.g., Fvs)that binds human LAG-3. “Lymphocyte-activation gene 3,” “LAG-3,” “LAG3,”“CD223,” and “duster of differentiation 3,” (e.g., Genebank AccessionNumbers NM_002286 and NP_002277 (human)) as used herein is meant amember of an immunoglobulin (Ig) superfamily, that is a type Itransmembrane protein with four extracellular Ig-like domains. LAG-3 isa negative regulator of T cells and appears to work in concert withother checkpoint molecules, including CTLA-4 and PD-1. In 1990, Triebelet al. discovered LAG3 as a transmembrane protein expressed by activatedhuman natural killer (NK) and T-cell lines, although it can also beexpressed on B cells. The human LAG3 gene has 20% identity with thehuman CD4 gene, resulting in surface-expressed LAG-3 protein being ableto bind major histocompatibility complex (MHC) class II molecules withhigh affinity. LAG-3 either exists as a cell surface dimer or as asoluble form released by interferon-gamma-producing CD4-positive T cellsvia proteolytic cleavage of a membrane-proximal connecting peptide.Exemplary LAG-3 sequences are depicted in FIG. 3.

This Fv, or anti-LAG-3 component (the anti-LAG-3 antigen binding domainor LAG-3 ABD) of the subject heterodimer fusion proteins is generally aset of 6 CDRs and/or a variable heavy domain and a variable light domainthat form an Fv domain that can bind human LAG-3. As described herein,there are a number of different formats that can be used, generallyeither by using a scFv or a Fab as outlined herein.

In certain embodiments, the ABDs of the invention comprise a heavy chainvariable region with frameworks from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene. For example, suchABDs may comprise or consist of a human ABD comprising heavy or lightchain variable regions that are “the product of” or “derived from” aparticular germline sequence. An ABD that is “the product of” or“derived from” a human germline immunoglobulin sequence can beidentified as such by comparing the amino acid sequence of the ABD tothe amino acid sequences of human germline immunoglobulins and selectingthe human germline immunoglobulin sequence that is closest in sequence(i.e., greatest % identity) to the sequence of the ABD. An ABD that is“the product of” or “derived from” a particular human germlineimmunoglobulin sequence may contain amino acid differences as comparedto the germline sequence, due to, for example, CDRs, naturally-occurringsomatic mutations or intentional introduction of site-directed mutation.However, a humanized ABD typically is at least 90% identical in aminoacids sequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify theABD as being derived from human sequences when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a humanized ABD may be at least95, 96, 97, 98 or 99%, or even at least 96%, 97%, 98%, or 99% identicalin amino acid sequence to the amino acid sequence encoded by thegermline immunoglobulin gene. Typically, a humanized ABD derived from aparticular human germline sequence will display no more than 10-20 aminoacid differences from the amino acid sequence encoded by the humangermline immunoglobulin gene (prior to the introduction of any skew, pIand ablation variants herein; that is, the number of variants isgenerally low, prior to the introduction of the variants of theinvention). In certain cases, the humanized ABD may display no more than5, or even no more than 4, 3, 2, or 1 amino acid difference from theamino acid sequence encoded by the germline immunoglobulin gene (again,prior to the introduction of any skew, pI and ablation variants herein;that is, the number of variants is generally low, prior to theintroduction of the variants of the invention). In one embodiment, theparent ABD has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

As shown herein, the anti-LAG-3 ABD can be in the form of either a Fabor an scFv.

In some embodiments, for example as depicted in FIGS. 21B and C, theanti-LAG-3 ABD is a scFv, wherein the VH and VL domains are joined usingan scFv linker, which can be optionally a charged scFv linker. As willbe appreciated by those in the art, the scFv can be assembled from N- toC-terminus, as N-VH-scFv linker-VL-C or as N-VL-scFv linker-VH-C, withthe C terminus of the scFv domain generally being linked to thehinge-CH2-CH3 Fc domain, wherein the hinge in this case serving as adomain linker. Suitable Fvs (including CDR sets and variableheavy/variable light domains) can be used in scFv formats or Fab formatsare shown in the Figures as well as disclosed in WO2017/218707, thecontents are hereby incorporated in its entirety for all purposes, andin particular for the LAG-3 ABDs in FIG. 11, the data in FIG. 18, FIG.55, FIG. 56, FIG. 63 and SEQ ID NO:s 36819-36962, SEQ ID NO:s35417-35606, SEQ ID NO:s 25914-32793 and SEQ ID NO:s 32794-33002sequences in the sequence listing.

As will further be appreciated by those in the art, all or part of thehinge (which can also be a wild type hinge from IgG1, IgG2 or IgG4 or avariant thereof, such as the IgG4 S241P or S228P hinge variant with thesubstitution proline at position 228 relative to the parent IgG4 hingepolypeptide (wherein the numbering S228P is according to the EU indexand the S241P is the Kabat numbering)) can be used as the domain linkerbetween the scFv and the CH2-CH3 domain, or a different domain linkersuch as depicted in the Figures can be used.

Alternatively, the LAG-3 ABD can be in the form of a Fab fragment. Inthis embodiment, the ABD is made up of a variable heavy domain,contributed by a heavy chain, and a variable light domain, contributedby a light chain. Suitable Fvs (including CDR sets and variableheavy/variable light domains) can be used in scFv formats or Fab formatsare shown in the Figures as well as disclosed in in WO2017/218707, thecontents are hereby incorporated in its entirety for all purposes, andin particular for the LAG-3 ABDs in FIG. 11, the data in FIG. 18, FIG.55, FIG. 56, FIG. 63 and SEQ ID NO:s 36819-36962, SEQ ID NO:s35417-35606, SEQ ID NO:s 25914-32793 and SEQ ID NO:s 32794-33002sequences in the sequence listing.

As will be appreciated by those in the art, suitable LAG-3 bindingdomains can comprise a set of 6 CDRs as depicted in the sequence listingand figures (e.g., FIGS. 12 and 13), either as they areunderlined/bolded or, in the case where a different numbering scheme isused as described herein and as shown in Table 2, as the CDRs that areidentified using other alignments within the variable heavy (VH) domainand variable light domain (VL) sequences of those depicted in thefigures (e.g., FIGS. 12 and 13A-C)and the sequence listing. SuitableLAG-3 ABDs that find use in the subject targeted IL-15/IL-15Rαheterodimeric fusion proteins can also include the entire VH and VLsequences as depicted in these sequences and figures, used as scFvs oras Fabs.

In one embodiment, the LAG-3 antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of any of the LAG-3 binding domainsdescribed in FIGS. 12 and 13A-C or the sequence listing.

In addition to the parental CDR sets disclosed in the figures andsequence listing that form an ABD to LAG-3, provided herein are variantLAG-3 ABDS having CDRs that include at least one modification of theLAG-3 ABD CDRs disclosed herein (e.g., FIGS. 12 and 13A-C). In oneembodiment, the heterodimeric fusion protein includes a LAG-3 ABD thatincludes a set of 6 CDRs with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidmodifications as compared to the 6 CDRs of a LAG-3 ABD as depicted inFIGS. 112 and 3A-C or the sequence listing. In certain embodiments, theLAG-3 ABD is capable of binding LAG-3 antigen, as measured by at leastone of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments.

In one embodiment, the LAG-3 ABD of the subject targeted IL-15/IL-15Rαheterodimeric fusion protein includes 6 CDRs that are at least 90, 95,97, 98 or 99% identical to the 6 CDRs of a LAG-3 ABD as depicted inFIGS. 12 and 13A-C or the sequence listing. In certain embodiments, theLAG-3 ABD is capable of binding to the LAG-3, as measured by at leastone of a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments.

In one embodiment of the subject targeted IL-15/IL-15Rα heterodimericfusion protein, the LAG-3 antigen binding domain includes the 6 CDRs(i.e., vhCDR1-3 and vlCDR1-3) of one of the following LAG-3 ABDs:7G8[LAG-3]_H₀_L0, 7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34.2A11[LAG-3]_H₀_L0, 2A11[LAG-3]_H1_L2, 2A11[LAG-3]_H1.44_L2.142,BMS-986016[LAG-3], IMP731[LAG-3], 13E2[LAG-3], 34F4[LAG-3],IMP761[LAG-3], H5L7[LAG-3], hu22D2[LAG-3], H4sH15482P[LAG-3],L35D4[LAG-3], L35G6[LAG-3], L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3],L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3](see, e.g., FIGS. 12 and 13A-C). In an exemplary embodiment, the LAG-3ABD is 7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD(see, e.g., FIG. 12).

In one embodiment, the LAG-3 antigen binding domain is a variant LAG-3antigen binding domain that includes 6 CDRs (i.e., vhCDR1-3 andvlCDR1-3), where the 6 CDRs include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10modifications as compared to the 6 CDRs of one of the following LAG-3ABDs: 7G8[LAG-3]_H₀_L0, 7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34.2A11[LAG-3]_H₀_L0, 2A11[LAG-3]_H1_L2, 2A11[LAG-3]_H1.44_L2.142,BMS-986016[LAG-3], IMP731[LAG-3], 13E2[LAG-3], 34F4[LAG-3],IMP761[LAG-3], H5L7[LAG-3], hu22D2[LAG-3], H4sH15482P[LAG-3],L35D4[LAG-3], L35G6[LAG-3], L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3],L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3](see, e.g., FIGS. 12 and 13A-C). In an exemplary embodiment, the LAG-3ABD is 7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD(see, e.g., FIG. 12).

In one embodiment, the LAG-3 antigen binding domain of the IL-15/IL-15Rαheterodimeric fusion protein is a variant LAG-3 antigen binding domainthat includes 6 CDRs (i.e., vhCDR1-3 and vlCDR1-3), where the 6 CDRs areat least 90, 95, 97, 98 or 99% identical as compared to the 6 CDRs) ofone of the following LAG-3 ABDs: 7G8[LAG-3]_H0_L0, 7G8[LAG-3]_H3_L1,7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H₀_L0, 2A11[LAG-3]_H1_L2,2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3], IMP731[LAG-3], 13E2[LAG-3],34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3], hu22D2[LAG-3],H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3], L33H11[LAG-3],L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3],L3C5[LAG-3], and L3E3[LAG-3] (see, e.g., FIGS. 12 and 13A-C). In anexemplary embodiment, the LAG-3 ABD is 7G8[LAG-3]_H3.30_L1.34 or2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG. 12).

In some embodiments, the LAG-3 ABD of the IL-15/IL-15Rα heterodimericfusion protein includes the variable heavy domain (VH) and variablelight domain (VL) of any of the LAG-ABDs disclosed herein, including,but not limited to those disclosed in FIGS. 12 and 13A-C. In addition tothe parental LAG-3 variable heavy and variable light domains disclosedherein, provided herein are subject targeted IL-15/IL-15Rα heterodimericfusion proteins having one or more LAG-3 ABDs that include a variableheavy domain and/or a variable light domain that are variants of a LAG-3ABD VH and VL domain disclosed herein. In one embodiment, the variant VHdomain and/or VL domain has from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 aminoacid changes from a VH and/or VL domain of a LAG-3 ABD depicted in FIGS.12, 13A-C, 29A and B, or the sequence listing. In certain embodiments,the LAG-3 ABD is capable of binding to LAG-3, as measured at least oneof a Biacore, surface plasmon resonance (SPR) and/or BLI (biolayerinterferometry, e.g., Octet assay) assay, with the latter findingparticular use in many embodiments.

In one embodiment, the variant VH and/or VL domain of the IL-15/IL-15Rαheterodimeric fusion protein is at least 90, 95, 97, 98 or 99% identicalto the VH and/or VL of a LAG-3 ABD as depicted in FIGS. 12 and 13A-C orthe sequence listing. In certain embodiments, the LAG-3 ABD is capableof binding to LAG-3, as measured by at least one of a Biacore, surfaceplasmon resonance (SPR) and/or BLI (biolayer interferometry, e.g., Octetassay) assay, with the latter finding particular use in manyembodiments.

In some embodiments, the LAG-3 ABD includes the VH and VL of one of thefollowing LAG-3 ABDs: 7G8[LAG-3]_H₀_L0, 7G8[LAG-3]_H3_L1,7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H₀_L0, 2A11[LAG-3]_H1_L2,2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3], IMP731[LAG-3], 13E2[LAG-3],34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3], hu22D2[LAG-3],H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3], L33H11[LAG-3],L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3], L3A1[LAG-3], L3A10[LAG-3],L3C5[LAG-3], and L3E3[LAG-3] (see, e.g., FIGS. 12 and 13A-C). In anexemplary embodiment, the LAG-3 ABD is 7G8[LAG-3]_H3.30_L1.34 or2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG. 12).

In some embodiments, the LAG-3 ABD includes a VH and VL, where the VHand/or VL includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidmodifications as compared to a VH and/or VL of a 7G8_H3.30_L1.34 or2A11_H1.144_L2.142 LAG-3 ABD (see, e.g., FIG. 12).

In certain embodiments, the LAG-3 ABD includes a VH and VL, where the VHand VL are at least 90, 95, 97, 98 or 99% identical as compared to a VHand VL of one of the following LAG-3 ABDs: 7G8[LAG-3]_H₀_L0,7G8[LAG-3]_H3_L1, 7G8[LAG-3]_H3.30_L1.34. 2A11[LAG-3]_H₀_L0,2A11[LAG-3]_H1_L2, 2A11[LAG-3]_H1.44_L2.142, BMS-986016[LAG-3],IMP731[LAG-3], 13E2[LAG-3], 34F4[LAG-3], IMP761[LAG-3], H5L7[LAG-3],hu22D2[LAG-3], H4sH15482P[LAG-3], L35D4[LAG-3], L35G6[LAG-3],L33H11[LAG-3], L32A9[LAG-3], L32D10[LAG-3], L32A4[LAG-3], L3A1[LAG-3],L3A10[LAG-3], L3C5[LAG-3], and L3E3[LAG-3] (see, e.g., FIGS. 12 and13A-C). In an exemplary embodiment, the LAG-3 ABD is7G8[LAG-3]_H3.30_L1.34 or 2A11[LAG-3]_H1.144_L2.142 LAG-3 ABD (see,e.g., FIG. 12).

C. Fc Domains

The Fc domain component of the invention is as described herein, whichgenerally contains skew variants and/or optional pI variants and/orablation variants are outlined herein. See for example the disclosure ofWO2017/218707 under the heading “IV Heterodimeric Antibodies”, includingsections IV.A, IV.B, IV.C, IV.D, IV.E, IV.F, IV.G, IV.H and IV.I, all ofwhich are expressly incorporated by reference in their entirety. Ofparticular use in the heterodimeric proteins of the present inventionare Fc domains containing “skew variants”, “pI variants”, “ablationvariants” and FcRn variants as outlined therein. Particularly usefulcombinations of such variants are depicted, for example, FIGS. 7A-F.

The Fc domains can be derived from IgG Fc domains, e.g., IgG1, IgG2,IgG3 or IgG4 Fc domains. In an exemplary embodiment, the subjectheterodimeric fusion protein provided herein includes an IgG1 Fc domain.The following describes Fc domains that are useful for IL-15/IL-15Rα Fcfusion monomers and anti-LAG-3 antibody fragments of the targetedIL-15/IL-15Rα heterodimeric fusion proteins.

Thus, the “Fc domain” includes the -CH2-CH3 domain, and optionally ahinge domain, and can be from human IgG1, IgG2, IgG3 or IgG4, with Fcdomains derived from IgG1. In some of the embodiments herein, when aprotein fragment, e.g., IL-15 or IL-15Rα is attached to an Fc domain, itis the C-terminus of the IL-15 or IL-15Rα construct that is attached toall or part of the hinge of the Fc domain. In other embodiments, when aprotein fragment, e.g., IL-15 or IL-15Rα, is attached to an Fc domain,it is the C-terminus of the IL-15 or IL-15Rα construct that is attachedto the CH1 domain of the Fc domain.

In some of the constructs and sequences outlined herein of an Fc domainprotein, the C-terminus of the IL-15 or IL-15Rα protein fragment isattached to the N-terminus of a domain linker, the C-terminus of whichis attached to the N-terminus of a constant Fc domain (N-IL-15 orIL-15Rα protein fragment-linker-Fc domain-C) although that can beswitched (N-Fc domain-linker-IL-15 or IL-15Rα protein fragment -C). Inother constructs and sequence outlined herein, C-terminus of a firstprotein fragment is attached to the N-terminus of a second proteinfragment, optionally via a domain linker, the C-terminus of the secondprotein fragment is attached to the N-terminus of a constant Fc domain,optionally via a domain linker. In yet other constructs and sequencesoutlined herein, a constant Fc domain that is not attached to a firstprotein fragment or a second protein fragment is provided. Aheterodimeric fusion protein can contain two or more of the exemplarymonomeric Fc domain proteins described herein. Any domain linker can beused to attach a IL-15 or IL-15Rα protein fragment to an Fc domain ofthe heterodimeric fusion protein provided herein. In some embodiments,the linker is any one of the linkers in FIG. 8.

In some embodiments, the linker is a “domain linker”, used to link anytwo domains (e.g., IL-15 or IL-15Rα protein fragment to Fc domain orscFv to Fc domain) as outlined herein together, some of which aredepicted in FIG. 8. While any suitable linker can be used, manyembodiments utilize a glycine-serine polymer, including for example(GS)n, (GSGGS)n (SEQ ID NO: 382), (GGGGS)n (SEQ ID NO: 14), and (GGGS)n(SEQ ID NO: 383), where n is an integer of at least one (and generallyfrom 1 to 2 to 3 to 4 to 5) as well as any peptide sequence that allowsfor recombinant attachment of the two domains with sufficient length andflexibility to allow each domain to retain its biological function. Insome cases, and with attention being paid to “strandedness”, as outlinedbelow, charged domain linkers.

In one embodiment, the heterodimeric fusion proteins contain at leasttwo constant domains which can be engineered to produce heterodimers,such as pI engineering. Other Fc domains that can be used includefragments that contain one or more of the CH1, CH2, CH3, and hingedomains of the invention that have been pI engineered. In particular,the formats depicted in FIG. 21 are heterodimeric fusion proteins,meaning that the protein has two associated Fc sequences self-assembledinto a heterodimeric Fc domain and at least one fusion protein (e.g., 1,2 or more fusion proteins) as more fully described below. In some cases,a first fusion protein is linked to a first Fc and a second fusionprotein is linked to a second Fc. In other cases, a first fusion proteinis linked to a first Fc, and the first fusion protein is non-covalentlyattached to a second fusion protein that is not linked to an Fc. In somecases, the heterodimeric fusion protein contains a first fusion proteinlinked to a second fusion protein which is linked a first Fc sequence,and a second Fc sequence that is not linked to either the first orsecond fusion proteins.

Accordingly, in some embodiments the present invention providesheterodimeric fusion proteins that rely on the use of two differentheavy chain variant Fc sequences, that will self-assemble to form aheterodimeric Fc domain fusion polypeptide.

The present invention is directed to novel constructs to provideheterodimeric fusion proteins that allow binding to one or more bindingpartners, ligands or receptors. The heterodimeric fusion constructs arebased on the self-assembling nature of the two Fc domains of the heavychains of antibodies, e.g., two “monomers” that assemble into a “dimer”.Heterodimeric Fc fusions are made by altering the amino acid sequence ofeach monomer as more fully discussed below. Thus, the present inventionis generally directed to the creation of heterodimeric fusion proteinswhich can co-engage binding partner(s) or ligand(s) or receptor(s) inseveral ways, relying on amino acid variants in the constant regionsthat are different on each chain to promote heterodimeric formationand/or allow for ease of purification of heterodimers over thehomodimers. Specific variants that are included in the Fc domains ofspecific embodiments of the subject heterodimeric fusion protein aredescribed in greater detail below.

1. Heterodimerization Variants

The present invention provides heterodimeric proteins, includingheterodimeric fusion proteins in a variety of formats. Suchheterodimeric proteins include two different Fc domains (one on each ofthe first and second monomers) that include modifications thatfacilitate the heterodimerization of the first and second monomersand/or allow for ease of purification of heterodimers over homodimers,collectively referred to herein as “heterodimerization variants.” Asdiscussed below, heterodimerization variants can include skew variants(e.g., the “knobs and holes” and “charge pairs” variants describedbelow) as well as “pI variants” that facilitates the separation ofhomodimers away from heterodimers. As is generally described in U.S.Pat. No. 9,605,084, hereby incorporated by reference in its entirety andspecifically as below for the discussion of heterodimerization variants,useful mechanisms for heterodimerization include “knobs and holes”(“KIH”) as described in U.S. Pat. No. 9,605,084, “electrostaticsteering” or “charge pairs” as described in U.S. Pat. No. 9,605,084, pIvariants as described in U.S. Pat. No. 9,605,084, and general additionalFc variants as outlined in U.S. Pat. No. 9,605,084 and below.

a. Skew Variants

In some embodiments, the subject heterodimeric protein includes skewvariants, which are one or more amino acid modifications in a first Fcdomain (A) and/or a second Fc domain (B) that favor the formation of Fcheterodimers (Fc dimers that include the first and the second Fc domain;A-B) over Fc homodimers (Fc dimers that include two of the first Fcdomain or two of the second Fc domain; A-A or B-B). Suitable skewvariants are included in the FIG. 29 of US Publ. App. No. 2016/0355608,hereby incorporated by reference in its entirety and specifically forits disclosure of skew variants, as well as in FIG. 4.

One mechanism for skew variants is generally referred to in the art as“knobs and holes,” referring to amino acid engineering that createssteric influences to favor heterodimeric formation and disfavorhomodimeric formation, as described in USSN 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety and specifically for the disclosure of“knobs and holes” mutations. This is sometime referred to herein as“steric variants.” The figures identify a number of “monomer A—monomerB” pairs that rely on “knobs and holes”. In addition, as described inMerchant et al., Nature Biotech. 16:677 (1998), these “knobs and holes”mutations can be combined with disulfide bonds to further favorformation of Fc heterodimers.

An additional mechanism for skew variants that finds use in thegeneration of heterodimers is sometimes referred to as “electrostaticsteering” as described in Gunasekaran et al., J. Biol. Chem.285(25):19637 (2010), hereby incorporated by reference in its entirety.This is sometimes referred to herein as “charge pairs.” In thisembodiment, electrostatics are used to skew the formation towardsheterodimerization. As those in the art will appreciate, these may alsohave an effect on pI, and thus on purification, and thus could in somecases also be considered pI variants. However, as these were generatedto force heterodimerization and were not used as purification tools,they are classified as “skew variants.” These include, but are notlimited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g., theseare “monomer” corresponding sets) and C220E/P228E/368E paired withC220R/E224R/P228R/K409R.

In some embodiments, the skew variants advantageously and simultaneouslyfavor heterodimerization based on both the “knobs and holes” mechanismas well as the “electrostatic steering” mechanisms described above. Insome embodiments, the heterodimeric protein includes one or more sets ofsuch heterodimerization skew variants. These variants come in “pairs” of“sets.” That is, one set of the pair is incorporated into the firstmonomer and the other set of the pair is incorporated into the secondmonomer. Exemplary “skew variants’ in this category includeS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L; K370S:S364K/E357Q; ora T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) “skew” variant amino acidsubstitution sets. In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers includes an Fcdomain that includes the amino acid substitutions S364K and E357Q andthe other monomer includes an Fc domain that includes the amino acidsubstitutions L368D and K370S; as above, the “strandedness” of thesepairs depends on the starting pI. It should be noted that these sets donot necessarily behave as “knobs in holes” variants, with a one-to-onecorrespondence between a residue on one monomer and a residue on theother. That is, these pairs of sets may instead form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25% homodimer A/A:50% heterodimer A/B:25%homodimer B/B).

In exemplary embodiments, the heterodimeric fusion protein includes aS364K/E357Q:L368D/K370S; L368D/K370S:S364K; L368E/K370S:S364K;T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L; K370S:S364K/E357Q; ora T366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) “skew” variant amino acidsubstitution set. In an exemplary embodiment, the heterodimeric fusionprotein includes a “S364K/E357Q:L368D/K370S” amino acid substitutionset.

In some embodiments, the skew variants provided herein are independentlyincorporated with other modifications, including, but not limited to,other skew variants (see, e.g., in FIG. 37 of US Publ. App. No.2012/0149876, herein incorporated by reference, particularly for itsdisclosure of skew variants), pI variants, isotypic variants, FcRnvariants, ablation variants, etc. into one or both of the first andsecond Fc domains of the heterodimeric fusion protein. Further,individual modifications can also independently and optionally beincluded or excluded from the subject heterodimeric fusion proteins.

b. pI (Isoelectric Point) Variants for Heterodimers

In some embodiments, the heterodimeric fusion protein includespurification variants that advantageously allow for the separation ofheterodimeric fusion proteins from homodimeric proteins (“pI variants”).

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

There are several basic mechanisms that can lead to ease of purifyingheterodimeric proteins. One such mechanism relies on the use of pIvariants which include one or more modifications that affect theisoelectric point of one or both of the monomers of the fusion protein,such that each monomer, and subsequently each dimeric species, has adifferent pI, thus allowing the isoelectric purification of A-A, A-B andB-B dimeric proteins. Alternatively, some formats also allow separationon the basis of size. As is further outlined above, it is also possibleto “skew” the formation of heterodimers over homodimers using skewvariants. Thus, a combination of heterodimerization skew variants and pIvariants find particular use in the subject heterodimeric fusionproteins provided herein.

Additionally, as more fully outlined below, depending on the format ofthe heterodimeric fusion protein, pI variants can be either containedwithin the constant region and/or Fc domains of a monomer, and/or domainlinkers can be used. In some embodiments, the heterodimeric fusionprotein includes additional modifications for alternativefunctionalities can also create pI changes, such as Fc, FcRn and KOvariants.

In the embodiments that utilizes pI as a separation mechanism to allowthe purification of heterodimeric fusion proteins, amino acidmodifications can be introduced into one or both of the monomers of theheterodimeric fusion protein. That is, the pI of one of the monomers(referred to herein for simplicity as “monomer A”) can be engineeredaway from monomer B, or both monomer A and B can be changed, with the pIof monomer A increasing and the pI of monomer B decreasing. Asdiscussed, the pI changes of either or both monomers can be done byremoving or adding a charged residue (e.g., a neutral amino acid isreplaced by a positively or negatively charged amino acid residue, e.g.,glutamine to glutamic acid), changing a charged residue from positive ornegative to the opposite charge (e.g., aspartic acid to lysine) orchanging a charged residue to a neutral residue (e.g., loss of a charge;lysine to serine.). A number of these variants are shown in the figures,including, FIGS. 4 and 5.

Creating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers can be done by usinga “wild type” heavy chain constant region and a variant region that hasbeen engineered to either increase or decrease its pI (wt A:B+ or wtA:B−), or by increasing one region and decreasing the other region(A+:B− or A−:B+).

Thus, in general, a component of some embodiments of the present subjectfusion proteins are amino acid variants in the Fc domains or constantdomain regions that are directed to altering the isoelectric point (pI)of at least one, if not both, of the monomers of a dimeric protein byincorporating amino acid substitutions (“pI variants” or “pIsubstitutions”) into one or both of the monomers. The separation of theheterodimers from the two homodimers can be accomplished if the pIs ofthe two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4and 0.5 or greater all finding use in the present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) of a heterodimeric fusion proteinto achieve good separation will depend in part on the starting pI of thecomponents. That is, to determine which monomer to engineer or in which“direction” (e.g., more positive or more negative), the sequences of theFc domains and any IL-15, IL-15Rα or linker included in each monomer arecalculated and a decision is made from there based on the pIs of themonomers. As is known in the art, different Fc domains, linkers IL-15,and IL-15Rα will have different starting pIs. In general, as outlinedherein, the pIs are engineered to result in a total pI difference ofeach monomer of at least about 0.1 logs, with 0.2 to 0.5 being preferredas outlined herein.

In general, as will be appreciated by those in the art, there are twogeneral categories of amino acid modifications that affect pI: thosethat increase the pI of the protein (basic changes) and those thatdecrease the pI of the protein (acidic changes). As described herein,all combinations of these variants can be used: one monomer may includea wild type Fc domain, or a variant Fc domain that does not display asignificantly different pI from wild-type, and the other monomerincludes a Fc domain that is either more basic or more acidic.Alternatively, each monomer may be changed, one to more basic and one tomore acidic.

In the case where pI variants are used to achieve heterodimerization, amore modular approach to designing and purifying heterodimeric fusionproteins is provided. Thus, in some embodiments, heterodimerizationvariants (including skew and pI variants) must be engineered. Inaddition, in some embodiments, the possibility of immunogenicityresulting from the pI variants is significantly reduced by importing pIvariants from different IgG isotypes such that pI is changed withoutintroducing significant immunogenicity (see isotypic variants below).Thus, an additional problem to be solved is the elucidation of low pIconstant domains with high human sequence content, e.g., theminimization or avoidance of non-human residues at any particularposition. Alternatively or in addition to isotypic substitutions, thepossibility of immunogenicity resulting from the pI variants issignificantly reduced by utilizing isosteric substitutions (e.g., Asn toAsp; and Gln to Glu).

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in US Publ. App. No. US 2012/0028304 (incorporated byreference in its entirety and specifically for the disclosure of pIvariants that provide additional function), lowering the pI of antibodyconstant domains (including those found in Fc fusions) can lead tolonger serum retention in vivo. These pI variants for increased serumhalf-life also facilitate pI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of Fc fusion proteins, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric fusion protein production isimportant.

Exemplary combinations of pI variants are shown in FIGS. 4 and 5, andFIG. 30 of US Publ. App. No. 2016/0355608, all of which are hereinincorporated by reference in its entirety and specifically for thedisclosure of pI variants. As outlined herein and shown in the figures,these changes are shown relative to IgG1, but all isotypes can bealtered this way, as well as isotype hybrids. In the case where theheavy chain constant domain is from IgG2-4, R133E and R133Q can also beused.

In some embodiments, modifications are made in the hinge of the Fcdomain, including positions 208, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pImutations and particularly substitutions can be made in one or more ofpositions 216-230, with 1, 2, 3, 4 or 5 mutations finding use. Again,all possible combinations are contemplated, alone or with other pIvariants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327,334 and 339, based on EU numbering. It should be noted that changes in233-236 can be made to increase effector function (along with 327A) inthe IgG2 backbone. Again, all possible combinations of these 14positions can be made; e.g., a heterodimeric fusion protein may includea variant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pIsubstitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, anon-native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non-native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the modifications can be independently andoptionally selected from position 355, 359, 362, 384, 389,392, 397, 418,419, 444 and 447 (EU numbering) of the CH3 region. Specificsubstitutions that find use in lowering the pI of CH3 domains include,but are not limited to, a non-native glutamine or glutamic acid atposition 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non-native glutamic acid at position 419, a non-nativeglutamic acid at position 359, a non-native glutamic acid at position362, a non-native glutamic acid at position 389, a non-native glutamicacid at position 418, a non-native glutamic acid at position 444, and adeletion or non-native aspartic acid at position 447. Exemplaryembodiments of pI variants are provided in FIG. 5.

In one embodiment, the heterodimeric fusion protein includes a monomerwith a variant Fc domain having pI variant modifications295E/384D/418E/421D (Q295E/N384D/Q418E/N421D when relative to humanIgG1). In one embodiment, the heterodimeric fusion protein includes amonomer with a variant Fc domain having pI variant modifications208D/295E/384D/418E/421D (N208D/Q295E/N384D/Q418E/N421D when relative tohuman IgG1). In some embodiments, the heterodimeric fusion proteinincludes a monomer with a variant Fc domain having pI variantmodifications 295E/384D/418E/421D (Q295E/N384D/Q418E/N421D when relativeto human IgG1). In one embodiment, the heterodimeric fusion proteinincludes a monomer with a variant Fc domain having pI variantmodifications 196K/199T/217R/228R/276K (Q196K/I199T/P217R/P228R/N276K)when relative to human IgG1).

In one embodiment, the heterodimeric fusion protein includes a monomerwith a variant Fc domain having pI variant modifications 217R/228R/276K(P217R/P228R/N276K when relative to human IgG1). Additional exemplary pIvariant modification that can be incorporated into the Fc domain of asubject are depicted in FIG. 5.

c. Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. A number of these areshown in FIG. 21 of US Publ. App. No. 2014/0370013, hereby incorporatedby reference. That is, IgG1 is a common isotype for therapeuticantibodies for a variety of reasons, including high effector function.However, the heavy constant region of IgG1 has a higher pI than that ofIgG2 (8.10 versus 7.31). By introducing IgG2 residues at particularpositions into the IgG1 backbone, the pI of the resulting monomer islowered (or increased) and additionally exhibits longer serum half-life.For example, IgG1 has a glycine (pI 5.97) at position 137, and IgG2 hasa glutamic acid (pI 3.22); importing the glutamic acid will affect thepI of the resulting protein. As is described below, a number of aminoacid substitutions are generally required to significant affect the pIof the variant Fc fusion protein. However, it should be noted asdiscussed below that even changes in IgG2 molecules allow for increasedserum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g., bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

d. Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the FIG. 19 ofUS2014/0370013. As discussed herein, which monomer to engineer isgenerally decided by the inherent pI of each monomer.

2. Additional Fc Variants for Additional Functionality

In addition to pI amino acid variants, there are a number of useful Fcamino acid modification that can be made for a variety of reasons,including, but not limited to, altering binding to one or more FcγRreceptors, altered binding to FcRn receptors, etc.

Accordingly, the proteins of the invention can include amino acidmodifications, including the heterodimerization variants outlinedherein, which includes the pI variants and steric variants. Each set ofvariants can be independently and optionally included or excluded fromany particular heterodimeric protein.

a. FcγR Variants

Accordingly, there are a number of useful Fc substitutions that can bemade to alter binding to one or more of the FcγR receptors.Substitutions that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa results in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell). Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), U.S. Ser. Nos. 11/174,287, 11/396,495,11/538,406, all of which are expressly incorporated herein by referencein their entirety and specifically for the variants disclosed therein.Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243A, 243L, 264A, 264V and299T.

In addition, amino acid substitutions that increase affinity for FcγRIIccan also be included in the Fc domain variants outlined herein. Thesubstitutions described in, for example, U.S. Ser. Nos. 11/124,620 and14/578,305 are useful.

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and increased serum half-life, asspecifically disclosed in U.S. Ser. No. 12/341,769, hereby incorporatedby reference in its entirety, including, but not limited to, 434S, 434A,428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L and 259I/308F/428L.

b. Ablation Variants

Similarly, another category of functional variants are “FcγR ablationvariants” or “Fc knock out (FcKO or KO)” variants. In these embodiments,for some therapeutic applications, it is desirable to reduce or removethe normal binding of the Fc domain to one or more or all of the Fcγreceptors (e.g., FcγR1, FcγRIIa, FcγRIIb, FcγRIIIa, etc.) to avoidadditional mechanisms of action. That is, for example, in manyembodiments, particularly in the use of bispecific immunomodulatoryantibodies desirable to ablate FcγRIIIa binding to eliminate orsignificantly reduce ADCC activity such that one of the Fc domainscomprises one or more Fcγ receptor ablation variants. These ablationvariants are depicted in FIG. 31 of U.S. Ser. No. 15/141,350, all ofwhich are herein incorporated by reference in its entirety, and each canbe independently and optionally included or excluded, with preferredaspects utilizing ablation variants selected from the group consistingof G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to the EU index. It should be noted that the ablation variantsreferenced herein ablate FcγR binding but generally not FcRn binding.

Exemplary ablation variants are provided in FIG. 5.

c. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants (including skew and/or pI variants) can beoptionally and independently combined in any way, as long as they retaintheir “strandedness” or “monomer partition”. In addition, all of thesevariants can be combined into any of the heterodimerization formats.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, skew and pI, arealso independently and optionally combined with Fc ablation variants, Fcvariants, FcRn variants, as generally outlined herein.

In addition, a monomeric Fc domain can comprise a set of amino acidsubstitutions that includes C220S/S267K/L368D/K370S orC220S/S267K/S364K/E357Q.

In addition, the heterodimeric fusion proteins can comprise skewvariants (e.g., a set of amino acid substitutions as shown in FIGS.1A-1C of U.S. Ser. No. 15/141,350, all of which are herein incorporatedby reference in its entirety), with particularly useful skew variantsbeing selected from the group consisting of S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L, K370S:S364K/E357Q, T366S/L368A/Y407V:T366W andT366S/L368A/Y407V/Y349C:T366W/S354C, optionally ablation variants,optionally charged domain linkers and the heavy chain comprises pIvariants.

In some embodiments, the Fc domain comprising an amino acid substitutionselected from the group consisting of: 236R, 239D, 239E, 243L, M252Y,V259I, 267D, 267E, 298A, V308F, 328F, 328R, 330L, 332D, 332E, M428L,N434A, N434S, 236R/328R, 239D/332E, M428L, 236R/328F, V259I/V308F,267E/328F, M428L/N434S, Y436I/M428L, Y436V/M428L, Y436I/N434S,Y436V/N434S, 239D/332E/330L, M252Y/S254T/T256E, V259I/V308F/M428L,E233P/L234V/L235A/G236del/S267K, G236R/L328R and PVA/S267K. In somecases, the Fc domain comprises the amino acid substitution 239D/332E. Inother cases, the Fc domain comprises the amino acid substitutionG236R/L328R or PVA/S267K.

In one embodiment, a particular combination of skew and pI variants thatfinds use in the present invention is T366S/L368A/Y407V:T366W(optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C) with one monomer comprisesQ295E/N384D/Q418E/N481D and the other a positively charged domainlinker. As will be appreciated in the art, the “knobs in holes” variantsdo not change pI, and thus can be used on either monomer. Usefulcombination of variants that can be used in particular formats of theinvention are included in FIGS. 7A-7F.

III. IL-15/IL-15Rα FC FUSION X LAG-3 ABD HETERODIMERIC PROTEINS

Provided herein are heterodimeric fusion proteins that can bind to thecheckpoint inhibitor LAG-3 antigen and can complex with the common gammachain (γc; CD132) and/or the Il-2 receptor β-chain (IL-2Rβ; CD122). Theheterodimeric fusion proteins can contain an IL-15/IL-15Rα-Fc fusionprotein and an antibody fusion protein. The IL-15/IL-15Rα-Fc fusionprotein can include as IL-15 protein (generally including amino acidsubstitutions) covalently attached to an IL-15Rα, and an Fc domain.Optionally, the IL-15 protein and IL-15Rα protein are noncovalentlyattached.

IV. USEFUL FORMATS OF THE INVENTION

As shown in FIG. 21, there are a number of useful formats of the subjecttargeted IL-15/IL-15Rα heterodimeric fusion proteins. In general, theheterodimeric fusion proteins provided herein have three functionalcomponents: an IL-15/IL-15Rα(sushi) component, an anti-LAG-3 component(also referred to as a “LAG-3 binding domain” or “LAG-3 antigen bindingdomain”), and an Fc component that includes a first Fc domain and secondFc domain, each of which can take different forms as outlined herein andeach of which can be combined with the other components in anyconfiguration.

The first and the second Fc domains can have a set of amino acid skewsubstitutions selected from the following skew variants: a)S267K/L368D/K370S:S267K/S364K/E357Q; b) S364K/E357Q:L368D/K370S; c)L368D/K370S:S364K; d) L368E/K370S:S364K; e) T411E/K360E/Q362E:D401K; f)L368D/K370S:S364K/E357L and g) K370S:S364K/E357Q according to EUnumbering. In an exemplary embodiment, the skew variants areS364K/E357Q:L368D/K370S.

In some embodiments, the first and/or the second Fc domains have anadditional set of pI amino acid substitutions selected from thefollowing pI variants: Q295E/N384D/Q418E/N421D,N208/Q295E/N384D/Q418E/N421D or Q196K/I199T/P217R/P228R/N276K, accordingto EU numbering.

Optionally, the first and/or the second Fc domains have an additionalset of ablation (“FcKO”) variants selected from the following FcKOvariants: G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

Optionally, the first and/or second Fc domains have 428L/434S variantsfor half-life extension.

In embodiments wherein a hinge or partial hinge is used to link an Fcdomain to a scFv, IL-15 or IL-15Rα domain, the hinge may optionalinclude a C220S substitution to prevent the hinge from formingundesirable disulfide bonds with any light chains.

Exemplary formats of the subject heterodimeric fusion proteins areprovided below.

A. scIL-15/Rα X scFv

One embodiment is shown in FIG. 21A, and comprises two monomers. Thefirst monomer comprises, from N- to C-terminus, the IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3 (with thesecond domain linker frequently being a hinge domain), and the secondmonomer comprises VH-scFv linker-VL-hinge-CH2-CH3 or VL-scFvlinker-VH-hinge-CH2-CH3, although in either orientation a domain linkercan be substituted for the hinge. This is generally referred to as“scIL-15/Rα X scFv”, with the “sc” standing for “single chain” referringto the attachment of the IL-15 variant and IL-15Rα(sushi) domain using acovalent linker. Preferred combinations of variants for this embodimentare found in FIGS. 21A and B.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X scFv” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first Fc domain; and b) a second monomer that includes,from N- to C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain. Any useful domain linker can be used toattach the various components of the heterodimeric protein including,but not limited to those in FIGS. 8 and 9A-C. In an exemplaryembodiment, the domain linkers that attach the IL-15 variant to thefirst Fc domain and the anti-LAG-3 scFv to the second Fc domain are eachantibody hinge domains.

In some embodiments, the anti-LAG-3 scFv includes a variable heavydomain (VH) covalently attached to a variable light domain (VL) by anscFv linker (e.g., FIGS. 9A-C). In one embodiment, the anti-LAG-3 scFvis from N- to C-terminus VH-scFv linker-VL. In another embodiment, theanti-LAG-3 scFv is from N- to C-terminus VL-scFv linker-VH. TheC-terminus of the anti-LAG-3 scFv is attached to the N terminus of thefirst Fc domain by a domain linker (e.g., an antibody hinge domain).

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIGS. 12 and 13A-C.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12. In one embodiment, the“scIL-15/Rα X scFv” format heterodimeric protein includes: a) a firstmonomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is afirst Fc domain; and b) a second monomer that includes, from N- toC-terminus, an anti -LAG-3 scFv-(hinge)-CH2-CH3, where CH2-CH3 is asecond Fc domain, and where the anti-LAG-3 scFv includes the variableheavy domain and variable light domain of 7G8_H3.30_L1.34. In oneembodiment, the “scIL-15/Rα X scFv” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where CH2-CH3 is a first Fc domain; and b) a second monomer thatincludes, from N- to C-terminus, an anti-LAG-3 scFv-(hinge)-CH2-CH3,where CH2-CH3 is a second Fc domain, and where the anti-LAG-3 scFvincludes the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the “scIL-15/Rα X scFv”format heterodimeric protein includes: a) a first monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b) asecond monomer that includes, from N- to C-terminus, anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain, and where theIL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the LAG-3 scFv includes theVH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15variant includes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the LAG-3scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the “scIL-15/Rα X scFv” format heterodimericprotein includes: a) a first monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(hinge)-CH2-CH3, where CH2-CH3 is a first Fc domain; and b) asecond monomer that includes, from N- to C-terminus, anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second Fc domain, where theanti-LAG-3 scFv includes the variable heavy domain and variable lightdomain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and the scFv includes the variableheavy and light domain pair of 7G8_H3.30_L1.34. In another embodiment ofthe scIL-15/Rα X scFv format heterodimeric protein, the IL-15 variantincludes amino acid substitutions N4D/N65D and the scFv includes thevariable heavy and light domain pair of 2A11_H1.144_L2.142. In oneembodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and the scFv includes the variable heavy and light domain pairof 7G8_H3.30_L1.34. In another embodiment of the scIL-15/Rα X scFvformat heterodimeric protein, the IL-15 variant includes amino acidsubstitutions D30N/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In yet another of embodiment,the IL-15 variant is the IL-15 D30N/E64Q/N65D variant and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “scIL-15/Rα X scFv”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; and b) a second monomer that includes, from N- toC-terminus, anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is asecond variant Fc domain, and where the first and second variant Fcdomains include the skew variant pair S364K/E357Q:L368D/K370S. In anexemplary embodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsL368D/K370S.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X scFv” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; and b) a second monomer thatincludes, from N- to C-terminus, anti-LAG-3 scFv-(domainlinker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain, where theIL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsL368D/K370S. In an exemplary embodiment, the LAG-3 scFv includes the VHand VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15variant includes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the LAG-3scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the “scIL-15/Rα X scFv”format heterodimeric protein includes: a) a first monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; andb) a second monomer that includes, from N- to C-terminus, anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc domain, wherethe anti-LAG-3 scFv includes the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34, and where the first and second variantFc domains include the skew variant pair S364K/E357Q:L368D/K370S. In oneembodiment, the “scIL-15/Rα X scFv” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where CH2-CH3 is a first Fc domain; and b) a second monomer thatincludes, from N- to C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3, whereCH2-CH3 is a second Fc domain, and where the anti-LAG-3 scFv includesthe variable heavy domain and variable light domain of2A11_H1.144_L2.142, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsL368D/K370S.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In oneembodiment, the “scIL-15/Rα X scFv” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where CH2-CH3 is a first variant Fc domain; and b) a second monomer thatincludes, from N- to C-terminus, anti-LAG-3 scFv-(hinge)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain, where the anti-LAG-3 scFvincludes the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In an exemplary embodiment, the firstvariant Fc domain includes skew variants L368D/K370S, and the secondvariant Fc domain includes skew variants L368D/K370S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment of the scIL-15/Rα X scFv format heterodimericprotein, the IL-15 variant includes amino acid substitutions D30N/N65Dand the scFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variant isthe IL-15 D30N/E64Q/N65D variant and the scFv includes the variableheavy and light domain pair of 7G8_H3.30_L1.34. In another embodiment,the IL-15 variant includes amino acid substitutions D30N/E64Q/N65D andthe scFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the “scIL-15/Rα X scFv” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; and b) asecond monomer that includes, from N- to C-terminus, anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc domain; wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain includes skew variants S364K/E357Q, where thefirst and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In some embodiments, the hinge of the firstand second monomers also each include amino acid substitution C220S. Incertain embodiments, the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In anexemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C. In an exemplary embodiment, the LAG-3 scFvincludes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-Cand the IL-15 variant includes amino acid substitutions N4D/N65D. Inanother exemplary embodiment, the LAG-3 scFv includes the VH and VL ofany of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions D30N/N65D. In yet another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/E64Q/N65D.

In the scIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21A format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the “scIL-15/Rα X scFv” format heterodimeric proteinincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; and b) asecond monomer that includes, from N- to C-terminus, anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a second variant Fc domain; wherethe anti-LAG-3 scFv includes the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the firstvariant Fc domain includes skew variants L368D/K370S and the secondvariant Fc domain includes skew variants S364K/E357Q, where the firstand second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In some embodiments, the hinge of the firstand second monomers also each include amino acid substitution C220S. Incertain embodiments, the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In aparticular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions N4D/N65D and the scFv includesthe variable heavy and light domain pair of 2A11_H1.144_L2.142. In oneembodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and the scFv includes the variable heavy and light domain pairof 7G8_H3.30_L1.34. In another embodiment of the scIL-15/Rα X scFvformat heterodimeric protein, the IL-15 variant includes amino acidsubstitutions D30N/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In yet another of embodiment,the IL-15 variant includes amino acid substitutions D30N/E64Q/N65D andthe scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and the scFv includes the variableheavy and light domain pair of 2A11_H1.144_L2.142.

B. scFv X ncIL-15/Rα

This embodiment is shown in FIG. 21B, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the IL-15Rα(sushi)domain-domain linker-CH2-CH3, and the second monomer comprises VH-scFvlinker-VL-hinge-CH2-CH3 or VL-scFv linker-VH-hinge-CH2-CH3, although ineither orientation a domain linker can be substituted for the hinge. Thethird monomer is the variant IL-15 domain. This is generally referred toas “ncIL-15/Rα X scFv” or “scFv X ncIL-15/Rα” with the “nc” standing for“non-covalent” referring to the self-assembling non-covalent attachmentof the IL-15 variant and IL-15Rα(sushi) domain.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; andc) an IL-15 variant, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex. Any useful domain linker can be used toattach the various components of the heterodimeric protein including,but not limited to those in FIGS. 8 and 9A-C. In an exemplaryembodiment, the domain linkers that attach the anti-LAG-3 scFv to thefirst Fc domain and the IL-15Rα(sushi) domain to the second Fc domainare each antibody hinge domains.

In some embodiments, the anti-LAG-3 scFv includes a variable heavydomain (VH) covalently attached to a variable light domain (VL) by anscFv linker (e.g., FIGS. 9A-C). In one embodiment, the anti-LAG-3 scFvis, from N- to C-terminus, VH-scFv linker-VL. In another embodiment, theanti-LAG-3 scFv is from, N- to C-terminus, VL-scFv linker-VH. TheC-terminus of the anti-LAG-3 scFv is attached to the N terminus of thefirst Fc domain by a domain linker (e.g., an antibody hinge domain).

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIGS. 12 and 13A-C.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; andc) an IL-15 variant, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, and where the anti-LAG-3 scFv includes thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; andc) an IL-15 variant, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, and where the anti-LAG-3 scFv includes thevariable heavy domain and variable light domain of 2A11_H1.144_L2.142.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “scFv X ncIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, whereCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; and c) an IL-15 variant, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, and wherethe IL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D,or D30N/E64Q/N65D. In an exemplary embodiment, the LAG-3 scFv includesthe VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions N4D/N65D. In anotherexemplary embodiment, the LAG-3 scFv includes the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/N65D. In yet another exemplary embodiment,the LAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS.12 and 13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scFv X ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, ananti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondFc domain; and c) an IL-15 variant, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where the anti-LAG-3 scFvincludes the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and the scFv includes the variableheavy and light domain pair of 7G8_H3.30_L1.34. In another embodiment,the IL-15 variant includes amino acid substitutions N4D/N65D and thescFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and the scFv includes the variable heavyand light domain pair of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 2A11_H1.144_L2.142.In yet another of embodiment, the IL-15 variant includes amino acidsubstitutions D30N/E64Q/N65D and the scFv includes the variable heavyand light domain pair of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/E64Q/N65D and thescFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “scFv X ncIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an anti-LAG-3 scFv-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain; and c) an IL-15 variant, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and c) an IL-15 variant, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In an exemplary embodiment, the LAG-3 scFv includes the VHand VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15variant includes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the LAG-3scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “scFv X ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 isa first variant Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and c) an IL-15 variant, wherethe IL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex,where the anti-LAG-3 scFv includes the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34, and where the first and secondvariant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “scFv X ncIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3,where CH2-CH3 is a second variant Fc domain; and c) an IL-15 variant,where the IL-15 variant and the IL-15Rα(sushi) domain form an IL-15complex, where the anti-LAG-3 scFv includes the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In anexemplary embodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsL368D/K370S. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scFv X ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, ananti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and c) an IL-15 variant, wherethe IL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex,where the anti-LAG-3 scFv includes the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe IL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D,or D30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and the scFv includes the variable heavy and light domain pairof 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and the scFv includesthe variable heavy and light domain pair of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and c) an IL-15 variant, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants L368D/K370S and the second variant Fcdomain includes skew variants S364K/E357Q, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In some embodiments, the hinge of the firstand second monomers also each include amino acid substitution C220S. Incertain embodiments, the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In anexemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C. In an exemplary embodiment, the LAG-3 scFvincludes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-Cand the IL-15 variant includes amino acid substitutions N4D/N65D. Inanother exemplary embodiment, the LAG-3 scFv includes the VH and VL ofany of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions D30N/N65D. In yet another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/E64Q/N65D.

In the ncIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pairof7G8_H3.30_L1. or the variable heavy and light domain pair2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21B format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and c) an IL-15 variant, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the anti-LAG-3scFv includes the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants L368D/K370S and the second variant Fc domainincludes skew variants S364K/E357Q, where the first and second variantFc domains each include FcKO variants E233P/L234V/L235A/G236del/S267K,where the first variant Fc domain includes pI variantsQ295E/N384D/Q418E/N421D, and where numbering is according to EUnumbering. In some embodiments, the hinge of the first and secondmonomers also each include amino acid substitution C220S. In certainembodiments, the first and second variant Fc domains each furtherinclude half-life extension variants M428L/N434S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment of the scIL-15/Rα X scFv format heterodimericprotein, the IL-15 variant includes amino acid substitutions D30N/N65Dand the scFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and the scFv includesthe variable heavy and light domain pair of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

C. scFv X dsIL-15/Rα

This embodiment is shown in FIG. 21C, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the IL-15Rα(sushi)domain-domain linker-CH2-CH3, wherein the IL-15Rα(sushi) domain has anengineered cysteine residue and the second monomer comprises VH-scFvlinker-VL-hinge-CH2-CH3 or VL-scFv linker-VH-hinge-CH2-CH3, although ineither orientation a domain linker can be substituted for the hinge. Thethird monomer is the variant IL-15 domain, also engineered to have acysteine variant amino acid, thus allowing a disulfide bridge to formbetween the IL-15Rα(sushi) domain and the variant IL-15 domain. This isgenerally referred to as “scFv X dsIL-15/Rα” or “dsIL-15/Rα X scFv”,with the “ds” standing for “disulfide”.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; and c) an IL-15 variant that includes an amino acidsubstitution for a cysteine residue, and where the cysteine residue onthe IL-15 variant and the cysteine residue on the IL-15Rα(sushi) domainform a disulfide bond. Any useful domain linker can be used to attachthe various components of the heterodimeric protein including, but notlimited to those in FIGS. 8 and 9A-C. In an exemplary embodiment, thedomain linkers that attach the anti-LAG-3 scFv to the first Fc domainand the IL-15Rα(sushi) domain to the second Fc domain and are eachantibody hinge domains.

Any useful domain linker can be used to attach the various components ofthe heterodimeric protein including, but not limited to those in FIGS. 8and 9A-C. In an exemplary embodiment, the domain linkers that attach theanti-LAG-3 scFv to the first Fc domain and the IL-15Rα(sushi) domain tothe second Fc domain and are each antibody hinge domains (e.g., anantibody hinge domain).

In some embodiments, the anti-LAG-3 scFv includes a variable heavydomain (VH) covalently attached to a variable light domain (VL) by anscFv linker (e.g., FIGS. 9A-C). In one embodiment, the anti-LAG-3 scFvis from N- to C-terminus VH-scFv linker-VL. In another embodiment, theanti-LAG-3 scFv is from N- to C-terminus VL-scFv linker-VH. TheC-terminus of the anti-LAG-3 scFv is attached to the N terminus of thefirst Fc domain by a domain linker (e.g., an antibody hinge domain).

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIGS. 12 and 13A-C.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; and c) an IL-15 variant that includes an amino acidsubstitution for a cysteine residue, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, and where the anti-LAG-3 scFv includes the variableheavy domain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an “scFvX dsIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, an anti-LAG-3 scFv-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second Fc domain and theIL-15Rα(sushi) domain includes an amino acid substitution for a cysteineresidue; and c) an IL-15 variant that includes an amino acidsubstitution for a cysteine residue, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, and where the anti-LAG-3 scFv includes the variableheavy domain and variable light domain of 2A11_H1.144_L2.142.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes theIL-15 N4D/N65D variant or the IL-15 D30N/N65D variant or the IL-15D30N/E64Q/N65D variant, as well as appropriate cysteine substitutions.In on embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“scFv X dsIL-15/Reα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; and c) an IL-15 variant that includes an amino acidsubstitution for a cysteine residue, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the LAG-3 scFvincludes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-Cand the IL-15 variant includes amino acid substitutions D30N/N65D. Inyet another exemplary embodiment, the LAG-3 scFv includes the VH and VLof any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant, as well as appropriate cysteine substitutions. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an “scFvX dsIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, an anti-LAG-3 scFv-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second Fc domain and theIL-15Rα(sushi) domain includes an amino acid substitution for a cysteineresidue; and c) an IL-15 variant that includes an amino acidsubstitution for a cysteine residue, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, where the anti-LAG-3 scFv includes the variable heavydomain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and the scFv includes the variable heavy and light domain pairof 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and the scFv includesthe variable heavy and light domain pair of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “scFv X dsIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an anti-LAG-3 scFv-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain and the IL-15Rα(sushi) domain includes an amino acid substitutionfor a cysteine residue; and c) an IL-15 variant that includes an aminoacid substitution for a cysteine residue, where the cysteine residue onthe IL-15 variant and the cysteine residue on the IL-15Rα(sushi) domainform a disulfide bond, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain and the IL-15Rα(sushi) domain includes an amino acidsubstitution for a cysteine residue; and c) an IL-15 variant thatincludes an amino acid substitution for a cysteine residue, where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In an exemplary embodiment, the LAG-3 scFv includes the VHand VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15variant includes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the LAG-3 scFv includes the VH and VL of any of the LAG-3ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the LAG-3scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “scFv X dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, where CH2-CH3 isa first variant Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain and the IL-15Rα(sushi) domainincludes an amino acid substitution for a cysteine residue; and c) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the cysteine residue on the IL-15 variant and thecysteine residue on the IL-15Rα(sushi) domain form a disulfide bond,where the anti-LAG-3 scFv includes the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34, and where the first and secondvariant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “scFv X dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, an anti-LAG-3 scFv-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3,where CH2-CH3 is a second variant Fc domain and the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue; andc) an IL-15 variant that includes an amino acid substitution for acysteine residue, where the cysteine residue on the IL-15 variant andthe cysteine residue on the IL-15Rα(sushi) domain form a disulfide bond,where the anti-LAG-3 scFv includes the variable heavy domain andvariable light domain of 2A11_H1.144_L2.142, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant with theappropriate cysteine substitutions.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain and the IL-15Rα(sushi) domain includes an amino acidsubstitution for a cysteine residue; and c) an IL-15 variant thatincludes an amino acid substitution for a cysteine residue, where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the anti-LAG-3 scFvincludes the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In an exemplary embodiment, the firstvariant Fc domain includes skew variants L368D/K370S, and the secondvariant Fc domain includes skew variants S364K/E357Q. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and the scFv includes the variable heavy and light domain pairof 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and the scFv includesthe variable heavy and light domain pair of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain and the IL-15Rα(sushi) domain includes an amino acid substitutionfor a cysteine residue; and c) an IL-15 variant that includes an aminoacid substitution for a cysteine residue, where the cysteine residue onthe IL-15 variant and the cysteine residue on the IL-15Rα(sushi) domainform a disulfide bond, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q, where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where the firstvariant Fc domain includes pI variants Q295E/N384D/Q418E/N421D, andwhere numbering is according to EU numbering. In some embodiments, thehinge of the first monomer and second monomer also each include aminoacid substitution C220S. In certain embodiments, the first and secondvariant Fc domains each further include half-life extension variantsM428L/N434S. In an exemplary embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In anexemplary embodiment, the LAG-3 scFv includes the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C. In an exemplary embodiment, theLAG-3 scFv includes the VH and VL of any of the LAG-3 ABDs in FIGS. 12and 13A-C and the IL-15 variant includes amino acid substitutionsN4D/N65D. In another exemplary embodiment, the LAG-3 scFv includes theVH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15variant includes amino acid substitutions D30N/N65D. In yet anotherexemplary embodiment, the LAG-3 scFv includes the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the dsIL-15/Rα X scFv format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21C format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scFv X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an anti-LAG-3scFv-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain and the IL-15Rα(sushi) domain includes an amino acid substitutionfor a cysteine residue; and c) an IL-15 variant that includes an aminoacid substitution for a cysteine residue, where the cysteine residue onthe IL-15 variant and the cysteine residue on the IL-15Rα(sushi) domainform a disulfide bond, where the anti-LAG-3 scFv includes the variableheavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q, where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where the firstvariant Fc domain includes pI variants Q295E/N384D/Q418E/N421D, andwhere numbering is according to EU numbering. In some embodiments, thehinge of the first monomer and second monomer also each include aminoacid substitution C220S. In certain embodiments, the first and secondvariant Fc domains each further include half-life extension variantsM428L/N434S. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and the scFv includes the variableheavy and light domain pair of 7G8_H3.30_L1.34. In another embodiment,the IL-15 variant includes amino acid substitutions N4D/N65D and thescFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and the scFv includes the variable heavyand light domain pair of 7G8_H3.30_L1.34. In another embodiment of thescIL-15/Rα X scFv format heterodimeric protein, the IL-15 variantincludes amino acid substitutions D30N/N65D and the scFv includes thevariable heavy and light domain pair of 2A11_H1.144_L2.142. In yetanother of embodiment, the IL-15 variant includes amino acidsubstitutions D30N/E64Q/N65D and the scFv includes the variable heavyand light domain pair of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/E64Q/N65D and thescFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142.

D. scIL-15/Rα X Fab

This embodiment is shown in FIG. 21D, and comprises three monomers. Thefirst monomer comprises, from N- to C-terminus, the IL-15Rα(sushi)domain-(domain linker)-variant IL-15-domain linker-CH2-CH3 and thesecond monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The thirdmonomer is a light chain, VL-CL. This is generally referred to as“scIL-15/Rα X Fab”, with the “sc” standing for “single chain”. ThescIL-15/Rα x Fab format (see FIG. 21 FIG. 21D) comprises IL-15Rα(sushi)fused to a variant IL-15 by a variable length linker (termed“scIL-15/Rα”) which is then fused to the N-terminus of a heterodimericFc-region (inclusive of the hinge). The second monomer is a heavy chain,VH-CH1-hinge-CH2-CH3, while a corresponding light chain (the thirdmonomer) is transfected separately so as to form a Fab with the VH.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavydomain and CH2-CH3 is a second Fc domain, and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain, where VH and VL form a LAG-3 binding domain. Any useful domainlinker can be used to attach the various components of the heterodimericprotein including, but not limited to those in FIGS. 8 and 9A-C. In anexemplary embodiment, the domain linkers that attach the IL-15 variantto the first Fc domain is an antibody hinge domain (e.g., an antibodyhinge domain).

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIGS. 12 and 13A-C.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric fusionprotein is an “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VHis a variable heavy domain and CH2-CH3 is a second Fc domain, and c) alight chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, and where VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively. In anotherembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavydomain and CH2-CH3 is a second Fc domain, and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain, and where VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142, respectively.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “scIL-15/Rα X Fab” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3 isa second Fc domain, and c) a light chain that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, wherein the VHand VL form a LAG-3 binding domain, and where the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In anexemplary embodiment, the VH and VL are the VH and VL of any of theLAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes aminoacid substitutions N4D/N65D. In another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VHis a variable heavy domain and CH2-CH3 is a second Fc domain, and c) alight chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, andwhere the IL-15 variant includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15variant includes amino acid substitutions N4D/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variantincludes amino acid substitutions D30N/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and VH and VL are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “scIL-15/Rα X Fab”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domainand CH2-CH3 is a second variant Fc domain, and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain, where VH and VL form a LAG-3 binding domain, and where the firstand second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variableheavy domain and CH2-CH3 is a second variant Fc domain, and c) a lightchain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, where VH and VL form a LAG-3 binding domain,where the IL-15 variant includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D, and where the first and second variant Fcdomains include the skew variant pair S364K/E357Q:L368D/K370S. In anexemplary embodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In an exemplary embodiment, the VH and VL are the VH and VLof any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, 2A11_H1.144_L2.142 thetargeted IL-15/IL-15Rα heterodimeric protein is an “scIL-15/Rα X Fab”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domainand CH2-CH3 is a second variant Fc domain, and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain, where VH and VL are the variable heavy domain and variable lightdomain of 7G8_H3.30_L1.34, respectively, and where the first and secondvariant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “scIL-15/Rα X Fab” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3 isa second variant Fc domain, and c) a light chain that includes from, N-to C-terminus, VL-VC, where VL is a variable light domain, where VH andVL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142, respectively, and where the first and second variantFc domains include the skew variant pair S364K/E357Q:L368D/K370S.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant here VH is avariable heavy domain and CH2-CH3 is a second variant Fc domain, and c)a light chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe IL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D,or D30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant here VH is avariable heavy domain and CH2-CH3 is a second variant Fc domain, and c)a light chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, where VH and VL form a LAG-3 binding domain,where the first variant Fc domain includes skew variants L368D/K370S andthe second variant Fc domain includes skew variants S364K/E357Q, wherethe first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In some embodiments, the hinge of the firstmonomer also includes amino acid substitution C220S. In certainembodiments, the first and second variant Fc domains each furtherinclude half-life extension variants M428L/N434S. In an exemplaryembodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, theVH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C. In an exemplary embodiment, the VH and VL are the VH and VL ofany of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the scIL-15/Rα X Fab format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21D format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “scIL-15/Rα X Fab” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, variant here VH is avariable heavy domain and CH2-CH3 is a second variant Fc domain, and c)a light chain that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain includes skew variants S364K/E357Q, where thefirst and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In some embodiments, the hinge of the firstmonomer also includes amino acid substitution C220S. In certainembodiments, the first and second variant Fc domains each furtherinclude half-life extension variants M428L/N434S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand the scFv includes the variable heavy and light domain pair of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 2A11_H1.144_L2.142. In one embodiment, the IL-15variant includes amino acid substitutions D30N/N65D and the scFvincludes the variable heavy and light domain pair of 7G8_H3.30_L1.34. Inanother embodiment of the scIL-15/Rα X scFv format heterodimericprotein, the IL-15 variant includes amino acid substitutions D30N/N65Dand the scFv includes the variable heavy and light domain pair of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and the scFv includesthe variable heavy and light domain pair of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and the scFv includes the variable heavy and light domainpair of 2A11_H1.144_L2.142.

E. Fab X ncIL-15/Rα

This embodiment is shown in FIG. 21E, and comprises four monomers. Thefirst monomer comprises, from N- to C-terminus, theIL-15Rα(sushi)domain-(domain linker)-CH2-CH3, and the second monomercomprises a heavy chain, VH-CH1-hinge-CH2-CH3. The third monomer is thelight chain that includes, from N-to C-terminus, a variable light domain(VL) and a light constant domain(CL). The fourth monomer is a variantIL-15 domain. This is generally referred to as “Fab X ncIL-15/Rα”, withthe “nc” standing for “non-covalent” referring to the self-assemblingnon-covalent attachment of the IL-15 variant and IL-15Rα(sushi)domain.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3 isa first Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; c) a third monomer that includes from, N-to C-terminus, VL-VC, where VL is a variable light domain, and d) afourth monomer comprising an IL-15 variant, where the VH and the VL forma LAG-3 binding domain, and where the IL-15 and IL-15Rα(sushi) domainform an IL-15 complex. Any useful domain linker can be used to attachthe various components of the heterodimeric protein including, but notlimited to those in FIGS. 8 and 9A-C. In an exemplary embodiment, thedomain linkers that attach the IL-15Rα(sushi) domain to the second Fcdomain is an antibody hinge domain (e.g., an antibody hinge domain).

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is a“Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c)a third monomer that includes from, N- to C-terminus, VL-VC, where VL isa variable light domain, and d) a fourth monomer comprising an IL-15variant, where the VH and the VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34, respectively, and where theIL-15 and IL-15Rα(sushi) domain form an IL-15 complex. In anotherembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an “FabX ncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c)a third monomer that includes from, N- to C-terminus, VL-VC, where VL isa variable light domain, and d) a fourth monomer comprising an IL-15variant, where the VH and the VL are the variable heavy domain andvariable light domain of 2A11_H1.144_L2.142, respectively, and where theIL-15 and IL-15Rα(sushi) domain form an IL-15 complex.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is a “Fab X ncIL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variableheavy domain and CH2-CH3 is a first Fc domain; b) a second monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second Fc domain; c) a third monomerthat includes from, N- to C-terminus, VL-VC, where VL is a variablelight domain, and d) a fourth monomer comprising an IL-15 variant, wherethe VH and the VL form a LAG-3 binding domain, where the IL-15 andIL-15Rα(sushi) domain form an IL-15 complex, and where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the VH andVL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is a “Fab X ncIL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3 isa first Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; c) a third monomer that includes from, N-to C-terminus, VL-VC, where VL is a variable light domain, and d) afourth monomer comprising an IL-15 variant, where the VH and the VL forma LAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domain forman IL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, andwhere the IL-15 variant includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15variant includes amino acid substitutions N4D/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variantincludes amino acid substitutions D30N/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and VH and VL are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “Fab XncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, and d) a fourthmonomer comprising an IL-15 variant, where the VH and the VL form aLAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domain form anIL-15 complex, and where the first and second variant Fc domains includethe skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain and CH2-CH3 isa first variant Fc domain; b) a second monomer that includes, from N- toC-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; c) a third monomer that includesfrom, N- to C-terminus, VL-VC, where VL is a variable light domain, andd) a fourth monomer comprising an IL-15 variant, where the VH and the VLform a LAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domainform an IL-15 complex, where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where thefirst and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q. In an exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutions N4D/N65D.In another exemplary embodiment, the VH and VL are the VH and VL of anyof the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/N65D. In yet another exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is a “Fab X ncIL-15/Rα”format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavy domainand CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain; c) a thirdmonomer that includes from, N- to C-terminus, VL-VC, where VL is avariable light domain, and d) a fourth monomer comprising an IL-15variant, where the VH and the VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34, respectively, where the IL-15and IL-15Rα(sushi) domain form an IL-15 complex and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “Fab X ncIL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variableheavy domain and CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain; c) a third monomer that includes from, N- to C-terminus, VL-VC,where VL is a variable light domain, and d) a fourth monomer comprisingan IL-15 variant, where the VH and the VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, where theIL-15 and IL-15Rα(sushi) domain form an IL-15 complex, and where thefirst and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is a“Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, and d) a fourthmonomer comprising an IL-15 variant, where the VH and the VL form aLAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domain form anIL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe IL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D,or D30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is a“Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, and d) a fourthmonomer comprising an IL-15 variant, where the VH and the VL form aLAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domain form anIL-15 complex, where the first variant Fc domain includes skew variantsL368D/K370S and the second variant Fc domain includes skew variantsS364K/E357Q, where the first and second variant Fc domains each includeFcKO variants E233P/L234V/L235A/G236del/S267K, where the hinge-firstvariant Fc domain of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, and where numbering is according to EUnumbering. In some embodiments, the hinge of the second monomer alsoincludes amino acid substitution C220S. In certain embodiments, thefirst and second variant Fc domains each further include half-lifeextension variants M428L/N434S. In an exemplary embodiment, the IL-15variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the VH andVL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the Fab X ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21E format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is a“Fab X ncIL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, and d) a fourthmonomer comprising an IL-15 variant, where the VH and the VL form aLAG-3 binding domain, where the IL-15 and IL-15Rα(sushi) domain form anIL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain includes skew variants S364K/E357Q, where thefirst and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI variants N208D/Q295E/N384D/Q418E/N421D,and where numbering is according to EU numbering. In some embodiments,the hinge of the second monomer also includes amino acid substitutionC220S. In certain embodiments, the first and second variant Fc domainseach further include half-life extension variants M428L/N434S. In aparticular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and the scFv includes the variable heavy andlight domain pair of 7G8_H3.30_L1.34. In a particular embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet another ofembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

F. Fab X dsIL-15/Rα

This embodiment is shown in FIG. 21F, and comprises four monomers. Thefirst monomer comprises, from N- to C-terminus, theIL-15Rα(sushi)domain-domain linker-CH2-CH3, wherein theIL-15Rα(sushi)domain has been engineered to contain a cysteine residue,and the second monomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3.The third monomer is a light chain that includes, from N-to C-terminus,a variable light domain (VL) and a constant light domain (CL). Thefourth monomer is the variant IL-15 domain, also engineered to have acysteine residue, such that a disulfide bridge is formed under nativecellular conditions. This is generally referred to as “Fab XdsIL-15/Rα”, with the “ds” standing for “disulfide”.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; c) a third monomer that includes, from N- toC-terminus, a VL-CL, where VL is a variable light domain; and d) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the VH and VL form a LAG-3 binding domain, and where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond. Any useful domain linkercan be used to attach the various components of the heterodimericprotein including, but not limited to those in FIGS. 8 and 9A-C. In anexemplary embodiment, the domain linkers that attach the IL-15Rα(sushi)domain to the second Fc domain is an antibody hinge domain (e.g., anantibody hinge domain).

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; c) a third monomer that includes, from N- toC-terminus, a VL-CL, where VL is a variable light domain; and d) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34, respectively, and where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond. In another embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “Fab X dsIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is avariable heavy domain and CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; c) a third monomer that includes, from N- toC-terminus, a VL-CL, where VL is a variable light domain; and d) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142, respectively, and where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D, with the appropriate cysteine amino acidsubstitutions. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “Fab X dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavy domain andCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain and the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue; c) a third monomer thatincludes, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain; and d) an IL-15 variant that includes an amino acid substitutionfor a cysteine residue, where the VH and VL form a LAG-3 binding domain,where the cysteine residue on the IL-15 variant and the cysteine residueon the IL-15Rα(sushi) domain form a disulfide bond, and where the IL-15variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the VH andVL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant, with the appropriate cysteine amino acid substitutions. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an “FabX dsIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereVH is a variable heavy domain and CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second Fc domain andthe IL-15Rα(sushi) domain includes an amino acid substitution for acysteine residue; c) a third monomer that includes, from N- toC-terminus, a VL-CL, where VL is a variable light domain; and d) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the VH and VL form a LAG-3 binding domain, where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet another ofembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “Fab X dsIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is avariable heavy domain and CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, an IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain and the IL-15Rα(sushi) domain includes an amino acid substitutionfor a cysteine residue; c) a third monomer that includes, from N- toC-terminus, a VL-CL, where VL is a variable light domain; and d) anIL-15 variant that includes an amino acid substitution for a cysteineresidue, where the VH and VL form a LAG-3 binding domain, where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain and the IL-15Rα(sushi) domain includes an amino acidsubstitution for a cysteine residue; c) a third monomer that includes,from N- to C-terminus, a VL-CL, where VL is a variable light domain; andd) an IL-15 variant that includes an amino acid substitution for acysteine residue, where the VH and VL form a LAG-3 binding domain, wherethe cysteine residue on the IL-15 variant and the cysteine residue onthe IL-15Rα(sushi) domain form a disulfide bond, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In an exemplary embodiment, the VH and VL are the VH and VLof any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “Fab X dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavy domain andCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, an IL-15Rα(sushi) domain-(domain linker)-CH2-CH3,where CH2-CH3 is a variant second Fc domain and the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue; c) athird monomer that includes, from N- to C-terminus, a VL-CL, where VL isa variable light domain; and d) an IL-15 variant that includes an aminoacid substitution for a cysteine residue, where the VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34,respectively, where the cysteine residue on the IL-15 variant and thecysteine residue on the IL-15Rα(sushi) domain form a disulfide bond, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “Fab X dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavydomain and CH2-CH3 is a first variant Fc domain; b) a second monomerthat includes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain and theIL-15Rα(sushi) domain includes an amino acid substitution for a cysteineresidue; c) a third monomer that includes, from N- to C-terminus, aVL-CL, where VL is a variable light domain; and d) an IL-15 variant thatincludes an amino acid substitution for a cysteine residue, where the VHand VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142, respectively, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant withappropriate cysteine substitutions. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “Fab X dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where VH is a variable heavydomain and CH2-CH3 is a first variant Fc domain; b) a second monomerthat includes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second variant Fc domain and theIL-15Rα(sushi) domain includes an amino acid substitution for a cysteineresidue; c) a third monomer that includes, from N- to C-terminus, aVL-CL, where VL is a variable light domain; and d) an IL-15 variant thatincludes an amino acid substitution for a cysteine residue, where the VHand VL form a LAG-3 binding domain, where the cysteine residue on theIL-15 variant and the cysteine residue on the IL-15Rα(sushi) domain forma disulfide bond, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe IL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D,or D30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain and the IL-15Rα(sushi) domain includes an amino acidsubstitution for a cysteine residue; c) a third monomer that includes,from N- to C-terminus, a VL-CL, where VL is a variable light domain; andd) an IL-15 variant that includes an amino acid substitution for acysteine residue, where the VH and VL form a LAG-3 binding domain, wherethe cysteine residue on the IL-15 variant and the cysteine residue onthe IL-15Rα(sushi) domain form a disulfide bond, where the first variantFc domain includes skew variants L368D/K370S and the second variant Fcdomain includes skew variants S364K/E357Q, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI variants N208D/Q295E/N384D/Q418E/N421D,and where numbering is according to EU numbering. In some embodiments,the hinge of the second monomer also includes amino acid substitutionC220S. In certain embodiments, the first and second variant Fc domainseach further include half-life extension variants M428L/N434S. In anexemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the Fab X dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21F format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “Fab X dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where VH is a variable heavy domain and CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain and the IL-15Rα(sushi) domain includes an amino acidsubstitution for a cysteine residue; c) a third monomer that includes,from N- to C-terminus, a VL-CL, where VL is a variable light domain; andd) an IL-15 variant that includes an amino acid substitution for acysteine residue, where the VH and VL form a LAG-3 binding domain, wherethe cysteine residue on the IL-15 variant and the cysteine residue onthe IL-15Rα(sushi) domain form a disulfide bond, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pI variantsN208D/Q295E/N384D/Q418E/N421D, and where numbering is according to EUnumbering. In some embodiments, the hinge of the second monomer alsoincludes amino acid substitution C220S. In certain embodiments, thefirst and second variant Fc domains each further include half-lifeextension variants M428L/N434S. In a particular embodiment, the IL-15variant includes amino acid substitutions N4D/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variantincludes amino acid substitutions D30N/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and VH and VL are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142.

G. mAb-scIL-15/Rα

This embodiment is shown in FIG. 21G, and comprises three monomers(although the fusion protein is a tetramer). The first monomer comprisesa heavy chain, VH-CH1-hinge-CH2-CH3. The second monomer comprises aheavy chain with a scIL-15 complex, VH-CH1-hinge-CH2-CH3-domainlinker-IL-15Rα(sushi)domain-domain linker-IL-15 variant. The third (andfourth) monomer are light chains, VL-CL. This is generally referred toas “mAb-scIL-15/Rα”, with the “sc” standing for “single chain”. Thisbinds the LAG-3 molecule bivalently.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-scIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fcdomain; and c) a third and fourth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the third monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefourth monomer form a second LAG-3 binding domain, and where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex. Any usefuldomain linker can be used to attach the various components of theheterodimeric protein including, but not limited to those in FIGS. 8 and9A-C.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-scIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second Fcdomain; and c) a third and fourth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the third monomer are the variableheavy domain and variable light domain of 7G8_H3.30_L1.34, respectively,where the VH of the second monomer and the VL of the fourth monomer arethe variable heavy domain and variable light domain of 7G8_H3.30_L1.34,respectively, and where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex. In another embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-scIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second Fc domain; and c) athird and fourth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, where theVH of the second monomer and the VL of the fourth monomer are thevariable heavy domain and variable light domain of 2A11_H1.144_L2.142,respectively, and where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes an IL-15variant that includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-scIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second Fc domain; and c) athird and fourth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, and where the IL-15variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the VH andVL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant, whereCH2-CH3 is a second Fc domain; and c) a third and fourth monomer thateach include, from N- to C-terminus, a VL-CL, where VL is a variablelight domain, where the VH of the first monomer and the VL of the thirdmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fourth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, andwhere the IL-15 variant includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In a particular embodiment, the IL-15variant includes amino acid substitutions N4D/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variantincludes amino acid substitutions D30N/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In yet another of embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and VH and VL are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-scIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second variantFc domain; and c) a third and fourth monomer that each include, from N-to C-terminus, a VL-CL, where VL is a variable light domain, where theVH of the first monomer and the VL of the third monomer form a firstLAG-3 binding domain, where the VH of the second monomer and the VL ofthe fourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, and wherethe first and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D and K370S, and the second variantFc domain includes skew variants S364K and E357Q. In an exemplaryembodiment, the first variant Fc domain includes skew variants S364K andE357Q, and the second variant Fc domain includes skew variants L368D andK370S.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-scIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant, whereCH2-CH3 is a second variant Fc domain; and c) a third and fourth monomerthat each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe third monomer form a first LAG-3 binding domain, where the VH of thesecond monomer and the VL of the fourth monomer form a second LAG-3binding domain, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where thefirst and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D and K370S, and the second variantFc domain includes skew variants S364K and E357Q. In an exemplaryembodiment, the first variant Fc domain includes skew variants S364K andE357Q, and the second variant Fc domain includes skew variants L368D andK370S. In an exemplary embodiment, the VH and VL are the VH and VL ofany of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-scIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively, where the VHof the second monomer and the VL of the fourth monomer are the variableheavy domain and variable light domain of 7G8_H3.30_L1.34, respectively,where the IL-15 variant and the IL-15Rα(sushi) domain form an IL-15complex, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, and where the first and second variant Fc domains includethe skew variant pair S364K/E357Q:L368D/K370S. In another embodiment,the targeted IL-15/IL-15Rα heterodimeric protein is an “mAb-scIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second variantFc domain; and c) a third and fourth monomer that each include, from N-to C-terminus, a VL-CL, where VL is a variable light domain, where theVH of the first monomer and the VL of the third monomer are the variableheavy domain and variable light domain of 2A11_H1.144_L2.142,respectively, where the VH of the second monomer and the VL of thefourth monomer are the variable heavy domain and variable light domainof 2A11_H1.144_L2.142, respectively, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D and K370S, and the second variantFc domain includes skew variants S364K and E357Q. In an exemplaryembodiment, the first variant Fc domain includes skew variants S364K andE357Q, and the second variant Fc domain includes skew variants L368D andK370S.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“mAb-scIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second variantFc domain; and c) a third and fourth monomer that each include, from N-to C-terminus, a VL-CL, where VL is a variable light domain, where theVH of the first monomer and the VL of the third monomer form a firstLAG-3 binding domain, where the VH of the second monomer and the VL ofthe fourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where VHand VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In an exemplary embodiment, the firstvariant Fc domain includes skew variants L368D and K370S, and the secondvariant Fc domain includes skew variants S364K and E357Q. In aparticular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another of embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142. In an exemplary embodiment, thefirst variant Fc domain includes skew variants L368D and K370S, and thesecond variant Fc domain includes skew variants S364K and E357Q. In anexemplary embodiment, the first variant Fc domain includes skew variantsS364K and E357Q, and the second variant Fc domain includes skew variantsL368D and K370S.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes the skewvariant set S364K/E357Q:L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K, theablation variants E233P/L234V/L235A/G236_/S267K on both first and secondmonomers, and optionally the 428L/434S variants on both first and secondmonomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-scIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant, whereCH2-CH3 is a second variant Fc domain; and c) a third and fourth monomerthat each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe third monomer form a first LAG-3 binding domain, where the VH of thesecond monomer and the VL of the fourth monomer form a second LAG-3binding domain, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions N208D/Q295E/N384D/Q418D/N421D and the hinge-second variantFc domain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants S364K/E357Q and the second variant Fcdomain include the skew variant pair L368D/K370S, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsQ196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc domain ofthe second monomer includes pI variants N208D/Q295E/N384D/Q418D/N421D,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-scIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second variantFc domain; and c) a third and fourth monomer that each include, from N-to C-terminus, a VL-CL, where VL is a variable light domain, where theVH of the first monomer and the VL of the third monomer form a firstLAG-3 binding domain, where the VH of the second monomer and the VL ofthe fourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where thefirst variant Fc domain includes skew variants S364K/E357Q and thesecond variant Fc domain include the skew variant pair L368D/K370S,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants S364K/E357Q and the second variant Fcdomain include the skew variant pair L368D/K370S, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the hinge-second variant Fc domain of the second monomer includes pIvariants N208D/Q295E/N384D/Q418D/N421D, and where numbering is accordingto EU numbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In an exemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the mAb-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21G format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K, theablation variants E233P/L234V/L235A/G236_/S267K on both first and secondmonomers, and optionally the 428L/434S variants on both first and secondmonomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-scIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant, whereCH2-CH3 is a second variant Fc domain; and c) a third and fourth monomerthat each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe third monomer form a first LAG-3 binding domain, where the VH of thesecond monomer and the VL of the fourth monomer form a second LAG-3binding domain, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain include the skew variant pair S364K/E357Q wherethe first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc domain ofthe second monomer includes pI variants Q196K/I199T/P271R/P228R/N276K,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-scIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant, where CH2-CH3 is a second variantFc domain; and c) a third and fourth monomer that each include, from N-to C-terminus, a VL-CL, where VL is a variable light domain, where theVH of the first monomer and the VL of the third monomer form a firstLAG-3 binding domain, where the VH of the second monomer and the VL ofthe fourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where VHand VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants L368D/K370S and the second variant Fc domaininclude the skew variant pair S364K/E357Q where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-second variant Fc domain of the second monomer includes pIvariants Q196K/I199T/P271R/P228R/N276K, and where numbering is accordingto EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-scIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second variantFc domain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-scIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-scIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant, where CH2-CH3 is a second variant Fc domain; andc) a third and fourth monomer that each include, from N- to C-terminus,a VL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the hinge-second variant Fc domain ofthe second monomer includes pI variants N208D/Q295E/N384D/Q418D/N421D,and where numbering is according to EU numbering. In certainembodiments, the first and second variant Fc domains each furtherinclude half-life extension variants M428L/N434S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet another ofembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

H. mAb-ncIL-15/Rα

This embodiment is shown in FIG. 21H, and comprises four monomers(although the heterodimeric fusion protein is a pentamer). The firstmonomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The secondmonomer comprises a heavy chain with an IL-15Rα(sushi) domain: e.g.,VH-CH1-hinge-CH2-CH3-domain linker-IL-15Rα(sushi) domain. The thirdmonomer is a variant IL-15 domain. The fourth (and fifth) monomer arelight chains, VL-CL. This is generally referred to as “mAb-ncIL-15/Rα”,with the “nc” standing for “non-covalent”. This also binds the LAG-3bivalently.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a thirdmonomer that includes an IL-15 variant; and d) a fourth and fifthmonomer that each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe fourth monomer form a first LAG-3 binding domain, where the VH ofthe second monomer and the VL of the fifth monomer form a second LAG-3binding domain, and where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex. Any useful domain linker can be used toattach the various components of the heterodimeric protein including,but not limited to those in FIGS. 8 and 9A-C.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a thirdmonomer that includes an IL-15 variant; and d) a fourth and fifthmonomer that each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe fourth monomer are the variable heavy domain and variable lightdomain of 7G8_H3.30_L1.34, respectively, where the VH of the secondmonomer and the VL of the fifth monomer are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively, and wherethe IL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex.In another embodiment, the targeted IL-15/IL-15Rα heterodimeric proteinis an “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second Fc domain; c) a thirdmonomer that includes an IL-15 variant; and d) a fourth and fifthmonomer that each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe fourth monomer are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142, respectively, where the VH of the secondmonomer and the VL of the fifth monomer are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, and wherethe IL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes an IL-15variant that includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second Fc domain; c) a third monomer that includes anIL-15 variant; and d) a fourth and fifth monomer that each include, fromN- to C-terminus, a VL-CL, where VL is a variable light domain, wherethe VH of the first monomer and the VL of the fourth monomer form afirst LAG-3 binding domain, where the VH of the second monomer and theVL of the fifth monomer form a second LAG-3 binding domain, where theIL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex, wherethe IL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex,and where the IL-15 variant includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In an exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions N4D/N65D. In anotherexemplary embodiment, the VH and VL are the VH and VL of any of theLAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes aminoacid substitutions D30N/N65D. In yet another exemplary embodiment, theVH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the IL-15 variantand the IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL arethe variable heavy domain and variable light domain of 7G8_H3.30_L1.34or 2A11_H1.144_L2.142, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-ncIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fifth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, and where the first and second variant Fcdomains include the skew variant pair S364K/E357Q:L368D/K370S. In anexemplary embodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In another exemplary embodiment, the first variant Fcdomain includes skew variants S364K/E357Q, and the second variant Fcdomain includes skew variants L368D/K370S.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes an IL-15 variant;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where theIL-15 variant includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In another exemplary embodiment, the first variant Fcdomain includes skew variants S364K/E357Q, and the second variant Fcdomain includes skew variants L368D/K370S. In an exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutions N4D/N65D.In another exemplary embodiment, the VH and VL are the VH and VL of anyof the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/N65D. In yet another exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer are the variable heavy domain and variable light domain of7G8_H3.30_L1.34, respectively, where the VH of the second monomer andthe VL of the fifth monomer are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34, respectively, where the IL-15 variantand the IL-15Rα(sushi) domain form an IL-15 complex, and where the firstand second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-ncIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142, respectively, where the VH of thesecond monomer and the VL of the fifth monomer are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142, respectively,where the IL-15 variant and the IL-15Rα(sushi) domain form an IL-15complex, and where the first and second variant Fc domains include theskew variant pair S364K/E357Q:L368D/K370S.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fifth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where thefirst and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q. In an exemplary embodiment,the first variant Fc domain includes skew variants S364K/E357Q, and thesecond variant Fc domain includes skew variants L368D/K370S. In aparticular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes the skewvariant set S364K/E357Q:L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K, theablation variants E233P/L234V/L235A/G236_/S267K on both first and secondmonomers, and optionally the 428L/434S variants on both first and secondmonomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes an IL-15 variant;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where theIL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex, wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain include the skew variant pair S364K/E357Q,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc domain ofthe second monomer includes pI variants Q196K/I199T/P271R/P228R/N276K,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-ncIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fifth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where the first variant Fcdomain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-second variant Fc domain of the second monomer includes pIvariants Q196K/I199T/P271R/P228R/N276K, and where numbering is accordingto EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the filth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second variantFc domain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-second variant Fc domain of the second monomer includes pIvariants N208D/Q295E/N384D/Q418D/N421D, and where numbering is accordingto EU numbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In an exemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the mAb-ncIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.21H format, the skew variant set S364K/E357Q:L368D/K370S, the pIvariants N208D/Q295E/N384D/Q418D/N421D and/orQ196K/I199T/P271R/P228R/N276K, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers. Inan exemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-ncIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes an IL-15 variant;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where theIL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex, whereVH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants L368D/K370S and the second variant Fc domaininclude the skew variant pair S364K/E357Q, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D and the hinge-second variant Fc domain ofthe second monomer includes pI variants Q196K/I199T/P271R/P228R/N276K,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-ncIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fifth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions N208D/Q295E/N384D/Q418D/N421D, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain include the skew variant pair S364K/E357Q,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-ncIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants S364K/E357Q and thesecond variant Fc domain include the skew variant pair L368D/K370S,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsQ196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc domain ofthe second monomer includes pI variants N208D/Q295E/N384D/Q418D/N421D,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-ncIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where CH2-CH3 is a second variant Fc domain; c)a third monomer that includes an IL-15 variant; and d) a fourth andfifth monomer that each include, from N- to C-terminus, a VL-CL, whereVL is a variable light domain, where the VH of the first monomer and theVL of the fourth monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fifth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-ncIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where CH2-CH3 is a second variant Fc domain; c) a third monomer thatincludes an IL-15 variant; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the IL-15 variant and the IL-15Rα(sushi) domain form anIL-15 complex, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, where VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, wherethe first variant Fc domain includes skew variants S364K/E357Q and thesecond variant Fc domain include the skew variant pair L368D/K370S,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In a particular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

I. mAb-dsIL-15/Rα

This embodiment is shown in FIG. 21I, and comprises four monomers(although the heterodimeric fusion protein is a pentamer). The firstmonomer comprises a heavy chain, VH-CH1-hinge-CH2-CH3. The secondmonomer comprises a heavy chain with an IL-15Rα(sushi) domain: e.g.,VH-CH1-hinge-CH2-CH3-domain linker-IL-15Rα(sushi) domain, where theIL-15Rα(sushi) domain has been engineered to contain a cysteine residue.The third monomer is a variant IL-15 domain, which has been engineeredto contain a cysteine residue, such that the IL-15 complex is formedunder physiological conditions. The fourth (and fifth) monomer are lightchains, VL-CL. This is generally referred to as “mAb-dsIL-15/Rα”, withthe “ds” standing for “disulfide”, and it binds LAG-3 bivalently.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, and where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond. Any useful domain linkercan be used to attach the various components of the heterodimericprotein including, but not limited to those in FIGS. 8 and 9A-C.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively, where the VHof the second monomer and the VL of the fifth monomer are the variableheavy domain and variable light domain of 7G8_H3.30_L1.34, respectively,and where the cysteine residue on the IL-15 variant and the cysteineresidue on the IL-15Rα(sushi) domain form a disulfide bond. In anotherembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, where theVH of the second monomer and the VL of the fifth monomer are thevariable heavy domain and variable light domain of 2A11_H1.144_L2.142,respectively, and where the cysteine residue on the IL-15 variant andthe cysteine residue on the IL-15Rα(sushi) domain form a disulfide bond.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes an IL-15variant that includes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, with the appropriate cysteine amino acid substitutions.In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, and where the IL-15 variant includes aminoacid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In anexemplary embodiment, the VH and VL are the VH and VL of any of theLAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes aminoacid substitutions N4D/N65D. In another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the mAb-dsIL-15/Rα format, one preferred embodiment 7G8_H3.30_L1.34or the variable heavy and light domain pair of 2A11_H1.144_L2.142 asshown in FIG. 12, with either the IL-15 N4D/N65D variant or the IL-15D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant, with theappropriate cysteine amino acid substitutions. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-dsIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, where VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes the skewvariant pair S364K/E357Q:L368D/K370S. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes an IL-15 variantthat includes an amino acid substitution for a cysteine residue; and d)a fourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, and where the first and second variant Fcdomains include the skew variant pair S364K/E357Q:L368D/K370S. In anexemplary embodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In another exemplary embodiment, the first variant Fcdomain includes skew variants S364K/E357Q, and the second variant Fcdomain includes skew variants L368D/K370S.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second variant Fc domain; c) a third monomer that includesan IL-15 variant that includes an amino acid substitution for a cysteineresidue; and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variantsL368D/K370S, and the second variant Fc domain includes skew variantsS364K/E357Q. In another exemplary embodiment, the first variant Fcdomain includes skew variants S364K/E357Q, and the second variant Fcdomain includes skew variants L368D/K370S. In an exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutions N4D/N65D.In another exemplary embodiment, the VH and VL are the VH and VL of anyof the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/N65D. In yet another exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where the IL-15Rα(sushi) domain includes an amino acid substitution fora cysteine residue and CH2-CH3 is a second variant Fc domain; c) a thirdmonomer that includes an IL-15 variant that includes an amino acidsubstitution for a cysteine residue; and d) a fourth and fifth monomerthat each include, from N- to C-terminus, a VL-CL, where VL is avariable light domain, where the VH of the first monomer and the VL ofthe fourth monomer are the variable heavy domain and variable lightdomain of 7G8_H3.30_L1.34, respectively, where the VH of the secondmonomer and the VL of the fifth monomer are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively, where thecysteine residue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondvariant Fc domain; c) a third monomer that includes an IL-15 variantthat includes an amino acid substitution for a cysteine residue; and d)a fourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, where theVH of the second monomer and the VL of the fifth monomer are thevariable heavy domain and variable light domain of 2A11_H1.144_L2.142,respectively, where the cysteine residue on the IL-15 variant and thecysteine residue on the IL-15Rα(sushi) domain form a disulfide bond, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant withappropriate cysteine substitutions. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “mAb-dsIL-15/Rα” formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where the IL-15Rα(sushi) domain includes an amino acid substitution fora cysteine residue and CH2-CH3 is a second Fc domain; c) a third monomerthat includes an IL-15 variant that includes an amino acid substitutionfor a cysteine residue; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain where the cysteine residue on the IL-15 variant and the cysteineresidue on the IL-15Rα(sushi) domain form a disulfide bond, where VH andVL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In an exemplary embodiment, the firstvariant Fc domain includes skew variants L368D/K370S, and the secondvariant Fc domain includes skew variants S364K/E357Q. In an exemplaryembodiment, the first variant Fc domain includes skew variantsS364K/E357Q, and the second variant Fc domain includes skew variantsL368D/K370S. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes the skewvariant set S364K/E357Q:L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K, theablation variants E233P/L234V/L235A/G236_/S267K on both first and secondmonomers, and optionally the 428L/434S variants on both first and secondmonomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, where the first variant Fc domain includesskew variants L368D/K370S and the second variant Fc domain include theskew variant pair S364K/E357Q, where the first and second variant Fcdomains each include FcKO variants E233P/L234V/L235A/G236del/S267K,where the hinge-first variant Fc domain of the first monomer includes pIsubstitutions N208D/Q295E/N384D/Q418D/N421D and the hinge-second variantFc domain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the first variant Fcdomain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the first variant Fcdomain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where the first variant Fcdomain includes skew variants S364K/E357Q and the second variant Fcdomain include the skew variant pair L368D/K370S, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-first variant Fc domainof the first monomer includes pI substitutionsQ196K/I199T/P271R/P228R/N276K and the hinge-second variant Fc domain ofthe second monomer includes pI variants N208D/Q295E/N384D/Q418D/N421D,and where numbering is according to EU numbering. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an “mAb-dsIL-15/Rα”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is afirst Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, where the first variant Fc domain includesskew variants S364K/E357Q and the second variant Fc domain include theskew variant pair L368D/K370S, where the first and second variant Fcdomains each include FcKO variants E233P/L234V/L235A/G236del/S267K,where the hinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where the IL-15Rα(sushi) domain includes an amino acid substitution fora cysteine residue and CH2-CH3 is a second Fc domain; c) a third monomerthat includes an IL-15 variant that includes an amino acid substitutionfor a cysteine residue; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the cysteine residue on the IL-15 variant and the cysteineresidue on the IL-15Rα(sushi) domain form a disulfide bond, where thefirst variant Fc domain includes skew variants S364K/E357Q and thesecond variant Fc domain include the skew variant pair L368D/K370S,where the first and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In an exemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the mAb-dsIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG. 21I format, theskew variant set S364K/E357Q:L368D/K370S, the pI variantsN208D/Q295E/N384D/Q418D/N421D and/or Q196K/I199T/P271R/P228R/N276K, theablation variants E233P/L234V/L235A/G236_/S267K on both first and secondmonomers, and optionally the 428L/434S variants on both first and secondmonomers. In an exemplary embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “mAb-dsIL-15/Rα” format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a first Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi)domain-(domain linker), where the IL-15Rα(sushi) domain includes anamino acid substitution for a cysteine residue and CH2-CH3 is a secondFc domain; c) a third monomer that includes an IL-15 variant thatincludes an amino acid substitution for a cysteine residue; and d) afourth and fifth monomer that each include, from N- to C-terminus, aVL-CL, where VL is a variable light domain, where the VH of the firstmonomer and the VL of the fourth monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fifthmonomer form a second LAG-3 binding domain, where the cysteine residueon the IL-15 variant and the cysteine residue on the IL-15Rα(sushi)domain form a disulfide bond, where VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions N208D/Q295E/N384D/Q418D/N421D and the hinge-second variantFc domain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions N208D/Q295E/N384D/Q418D/N421D, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where the IL-15Rα(sushi) domain includes an amino acid substitution fora cysteine residue and CH2-CH3 is a second Fc domain; c) a third monomerthat includes an IL-15 variant that includes an amino acid substitutionfor a cysteine residue; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the cysteine residue on the IL-15 variant and the cysteineresidue on the IL-15Rα(sushi) domain form a disulfide bond, where VH andVL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants L368D/K370S and the second variant Fc domaininclude the skew variant pair S364K/E357Q, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsQ196K/I199T/P271R/P228R/N276K, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K and the hinge-second variantFc domain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “mAb-dsIL-15/Rα” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3-(domainlinker)-IL-15Rα(sushi) domain-(domain linker), where the IL-15Rα(sushi)domain includes an amino acid substitution for a cysteine residue andCH2-CH3 is a second Fc domain; c) a third monomer that includes an IL-15variant that includes an amino acid substitution for a cysteine residue;and d) a fourth and fifth monomer that each include, from N- toC-terminus, a VL-CL, where VL is a variable light domain, where the VHof the first monomer and the VL of the fourth monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefifth monomer form a second LAG-3 binding domain, where the cysteineresidue on the IL-15 variant and the cysteine residue on theIL-15Rα(sushi) domain form a disulfide bond, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants S364K/E357Q and the second variant Fc domain include the skewvariant pair L368D/K370S, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thehinge-first variant Fc domain of the first monomer includes pIsubstitutions Q196K/I199T/P271R/P228R/N276K, and where numbering isaccording to EU numbering. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “mAb-dsIL-15/Rα” format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3-(domain linker)-IL-15Rα(sushi) domain-(domain linker),where the IL-15Rα(sushi) domain includes an amino acid substitution fora cysteine residue and CH2-CH3 is a second Fc domain; c) a third monomerthat includes an IL-15 variant that includes an amino acid substitutionfor a cysteine residue; and d) a fourth and fifth monomer that eachinclude, from N- to C-terminus, a VL-CL, where VL is a variable lightdomain, where the VH of the first monomer and the VL of the fourthmonomer form a first LAG-3 binding domain, where the VH of the secondmonomer and the VL of the fifth monomer form a second LAG-3 bindingdomain, where the cysteine residue on the IL-15 variant and the cysteineresidue on the IL-15Rα(sushi) domain form a disulfide bond, where VH andVL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants S364K/E357Q and the second variant Fc domaininclude the skew variant pair L368D/K370S, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the hinge-second variant Fcdomain of the second monomer includes pI variantsN208D/Q295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In a particular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

J. Central-IL-15/Rα

This embodiment is shown in FIG. 21J, and comprises four monomersforming a tetramer. The first monomer comprises a VH-CH1-[optionaldomain linker]-IL-15 variant-[optional domain linker]-CH2-CH3, with thesecond optional domain linker sometimes being the hinge domain. Thesecond monomer comprises a VH-CH1-[optional domainlinker]-IL-15Rα(sushi) domain-[optional domain linker]-CH2-CH3, with thesecond optional domain linker sometimes being the hinge domain. Thethird (and fourth) monomers are light chains, VL-CL. This is generallyreferred to as “central-IL-15/Rα”.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-IL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstFc domain; b) a second monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; and d) a third and fourth monomer thateach include from N-to C-terminus, a VL-CL, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, and where the IL-15 variantand the IL-15Rα(sushi) domain form an IL-15 complex. Any useful domainlinker can be used to attach the various components of the heterodimericprotein including, but not limited to those in FIGS. 8 and 9A-C. In anexemplary embodiment, the domain linkers that attach the IL-15 variantto the first Fc domain and the IL-15Rα(sushi) domain to the second Fcdomain are each antibody hinge domains.

In the central-IL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the central-IL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-IL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstFc domain; b) a second monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; and d) a third and fourth monomer thateach include from N-to C-terminus, a VL-CL, where the VH of the firstmonomer and the VL of the third monomer are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34, respectively, where the VHof the second monomer and the VL of the fourth monomer are the variableheavy domain and variable light domain of 7G8_H3.30_L1.34, respectively,and where the IL-15 variant and the IL-15Rα(sushi) domain form an IL-15complex. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “central-IL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3is a first Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a third andfourth monomer that each include from N-to C-terminus, a VL-CL, wherethe VH of the first monomer and the VL of the third monomer are thevariable heavy domain and variable light domain of 2A11_H1.144_L2.142,respectively, where the VH of the second monomer and the VL of thefourth monomer are the variable heavy domain and variable light domainof 2A11_H1.144_L2.142, respectively, and where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex.

In the “central-IL-15/Rα”format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “central-IL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 isa second Fc domain; and d) a third and fourth monomer that each includefrom N-to C-terminus, a VL-CL, where the VH of the first monomer and theVL of the third monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fourth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, and where the IL-15 variant includes aminoacid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In anexemplary embodiment, the VH and VL are the VH and VL of any of theLAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes aminoacid substitutions N4D/N65D. In another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the central-IL-15/Rαformat, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair7G8_H3.30_L1.34 or the variable heavy and light domain pair2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “central-IL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3is a first Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a third andfourth monomer that each include from N-to C-terminus, a VL-CL, wherethe VH of the first monomer and the VL of the third monomer form a firstLAG-3 binding domain, where the VH of the second monomer and the VL ofthe fourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where VHand VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the central-IL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an“central-IL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-(domain linker)-IL-15variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, where CH2-CH3 isa second Fc domain; and d) a third and fourth monomer that each includefrom N-to C-terminus, a VL-CL, where the VH of the first monomer and theVL of the third monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fourth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, and where the first and second variant Fcdomains include the skew variant pair S364K/E357Q:L368D/K370S. In anexemplary embodiment, the first variant Fc domain includes skew variantsS364K and E357Q, and the second variant Fc domain includes skew variantsL368D and K370S. In another exemplary embodiment, the first variant Fcdomain includes skew variants L368D and K370S, and the second variant Fcdomain includes skew variants S364K and E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-IL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstFc domain; b) a second monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second Fc domain; and d) a third and fourth monomer thateach include from N-to C-terminus, a VL-CL, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variants S364K andE357Q, and the second variant Fc domain includes skew variants L368D andK370S. In another exemplary embodiment, the first variant Fc domainincludes skew variants L368D and K370S, and the second variant Fc domainincludes skew variants S364K and E357Q. In an exemplary embodiment, theVH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutions N4D/N65D.In another exemplary embodiment, the VH and VL are the VH and VL of anyof the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/N65D. In yet another exemplary embodiment,the VH and VL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and13A-C and the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D.

In the central-IL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “central-IL-15/Rα”format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain; and d) a third and fourth monomer that each include from N-toC-terminus, a VL-CL, where the VH of the first monomer and the VL of thethird monomer are the variable heavy domain and variable light domain of7G8_H3.30_L1.34, respectively, where the VH of the second monomer andthe VL of the fourth monomer are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34, respectively, where the IL-15 variantand the IL-15Rα(sushi) domain form an IL-15 complex, and where the firstand second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “central-IL-15/Rα”format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3,where CH2-CH3 is a first variant Fc domain; b) a second monomer thatincludes, from N- to C-terminus, a VH-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-CH2-CH3, where CH2-CH3 is a second variant Fcdomain; and d) a third and fourth monomer that each include from N-toC-terminus, a VL-CL, where the VH of the first monomer and the VL of thethird monomer are the variable heavy domain and variable light domain of2A11_H1.144_L2.142, respectively, where the VH of the second monomer andthe VL of the fourth monomer are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142, respectively, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, and wherethe first and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S.

In the central-IL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/E64Q/N65D variant with appropriate cysteine substitutions. Inone embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“central-IL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-(domain linker)-IL-15variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first variant Fcdomain; b) a second monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and d) a third and fourth monomerthat each include from N- to C-terminus, a VL-CL, where the VH of thefirst monomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where thefirst and second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants S364K and E357Q, and the second variantFc domain includes skew variants L368D and K370S. In another exemplaryembodiment, the first variant Fc domain includes skew variants L368D andK370S, and the second variant Fc domain includes skew variants S364K andE357Q. In a particular embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D and VH and VL are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions N4D/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the central-IL-15/Rα format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-IL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variantFc domain; b) a second monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(hinge)-CH2-CH3, where CH2-CH3is a second variant Fc domain; and d) a third and fourth monomer thateach include from N-to C-terminus, a VL-CL, where the VH of the firstmonomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the first variantFc domain includes skew variants L368D/K370S and the second variant Fcdomain include the skew variant pair S364K/E357Q, where the first andsecond variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI substitutions Q295E/N384D/Q418D/N421D, and where numberingis according to EU numbering. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “central-IL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-(domain linker)-IL-15variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b)a second monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(hinge)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and d) a third and fourth monomer that each includefrom N-to C-terminus, a VL-CL, where the VH of the first monomer and theVL of the third monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fourth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where the first variant Fc domain includesskew variants S364K/E357Q and the second variant Fc domain include theskew variant pair L368D/K370S, where the first and second variant Fcdomains each include FcKO variants E233P/L234V/L235A/G236del/S267K,where the second variant Fc domain of the second monomer includes pIsubstitutions Q295E/N384D/Q418D/N421D, and where numbering is accordingto EU numbering. In certain embodiments, the first and second variant Fcdomains each further include half-life extension variants M428L/N434S.In an exemplary embodiment, the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the central-IL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.21K, the skew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers. Inone embodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“central-IL-15/Rα”format heterodimeric protein that includes: a) a firstmonomer that includes, from N- to C-terminus, a VH-(domain linker)-IL-15variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b)a second monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(hinge)-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and d) a third and fourth monomer that each includefrom N-to C-terminus, a VL-CL, where the VH of the first monomer and theVL of the third monomer form a first LAG-3 binding domain, where the VHof the second monomer and the VL of the fourth monomer form a secondLAG-3 binding domain, where the IL-15 variant and the IL-15Rα(sushi)domain form an IL-15 complex, where VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain include the skewvariant pair S364K/E357Q, where the first and second variant Fc domainseach include FcKO variants E233P/L234V/L235A/G236del/S267K, where thefirst variant Fc domain includes pI substitutionsQ295E/N384D/Q418D/N421D, and where numbering is according to EUnumbering. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “central-IL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is afirst variant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15Rα(sushi) domain-(hinge)-CH2-CH3,where CH2-CH3 is a second variant Fc domain; and d) a third and fourthmonomer that each include from N-to C-terminus, a VL-CL, where the VH ofthe first monomer and the VL of the third monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where VHand VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants S364K/E357Q and the second variant Fc domaininclude the skew variant pair L368D/K370S, where the first and secondvariant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the second variant Fc domain ofthe second monomer includes pI substitutions Q295E/N384D/Q418D/N421D,and where numbering is according to EU numbering. In certainembodiments, the first and second variant Fc domains each furtherinclude half-life extension variants M428L/N434S. In a particularembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

K. Central scIL-15/Rα

This embodiment is shown in FIG. 21K, and comprises four monomersforming a tetramer. The first monomer comprises a VH-CH1-[optionaldomain linker]-IL-15Rα(sushi) domain-domain linker-IL-15variant-[optional domain linker]-CH2-CH3, with the second optionaldomain linker sometimes being the hinge domain. The second monomercomprises a VH-CH1-hinge-CH2-CH3. The third (and fourth) monomers arelight chains, VL-CL. This is generally referred to as“central-scIL-15/Rα”, with the “sc” standing for “single chain”.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3is a second Fc domain; and d) a third and fourth monomer that eachinclude from N-to C-terminus, a VL-CL, where the VH of the first monomerand the VL of the third monomer form a first LAG-3 binding domain, wherethe VH of the second monomer and the VL of the fourth monomer form asecond LAG-3 binding domain, and where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex. Any useful domain linkercan be used to attach the various components of the heterodimericprotein including, but not limited to those in FIGS. 8 and 9A-C. In anexemplary embodiment, the domain linker that attaches the IL-15 variantto the first Fc domain is an antibody hinge domain.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having any of the variable heavy and light domain pairsas shown in FIG. 12.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair7G8_H3.30_L1.34 34 or the variable heavy and light domain pair2A11_H1.144_L2.142 as shown in as shown in FIG. 12.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b) a second monomerthat includes, from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3is a second Fc domain; and d) a third and fourth monomer that eachinclude from N-to C-terminus, a VL-CL, where the VH of the first monomerand the VL of the third monomer are the variable heavy domain andvariable light domain of 7G8_H3.30_L1.34, respectively, where the VH ofthe second monomer and the VL of the fourth monomer are the variableheavy domain and variable light domain of 7G8_H3.30_L1.34, respectively,and where the IL-15 variant and the IL-15Rα(sushi) domain form an IL-15complex. In another embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “central-scIL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-(domainlinker)-IL-15 variant-CH2-CH3, where CH2-CH3 is a first Fc domain; b) asecond monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a second Fc domain; and d) a third and fourth monomerthat each include from N-to C-terminus, a VL-CL, where the VH of thefirst monomer and the VL of the third monomer are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142, respectively,where the VH of the second monomer and the VL of the fourth monomer arethe variable heavy domain and variable light domain of2A11_H1.144_L2.142, respectively, and where the IL-15 variant and theIL-15Rα(sushi) domain form an IL-15 complex.

In the central-scIL-15/Rα format, one preferred embodiment utilizes anIL-15 variant that includes amino acid substitutions N4D/N65D,D30N/N65D, or D30N/E64Q/N65D. In one embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “central-scIL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first Fc domain; b) a second monomer that includes, from N-to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain;and d) a third and fourth monomer that each include from N-toC-terminus, a VL-CL, where the VH of the first monomer and the VL of thethird monomer form a first LAG-3 binding domain, where the VH of thesecond monomer and the VL of the fourth monomer form a second LAG-3binding domain, where the IL-15 variant and the IL-15Rα(sushi) domainform an IL-15 complex, and where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In an exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions N4D/N65D. In another exemplary embodiment, the VH and VLare the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and theIL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12, with either the IL-15 N4D/N65Dvariant or the IL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65Dvariant. In one embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “central-scIL-15/Rα”format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, aVH-(domain linker)-IL-15Rα(sushi) domain-(domain linker)-IL-15variant-(domain linker)-CH2-CH3, where CH2-CH3 is a first Fc domain; b)a second monomer that includes, from N- to C-terminus, aVH-hinge-CH2-CH3, where CH2-CH3 is a second Fc domain; and d) a thirdand fourth monomer that each include from N-to C-terminus, a VL-CL,where the VH of the first monomer and the VL of the third monomer form afirst LAG-3 binding domain, where the VH of the second monomer and theVL of the fourth monomer form a second LAG-3 binding domain, where theIL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex, whereVH and VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, and where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In a particular embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D and VH and VL are the variable heavydomain and variable light domain of 7G8_H3.30_L1.34. In anotherembodiment, the IL-15 variant includes amino acid substitutions N4D/N65Dand VH and VL are the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In one embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 7G8_H3.30_L1.34. In another embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142. In yet another embodiment, the IL-15 variantincludes amino acid substitutions D30N/E64Q/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 2A11_H1.144_L2.142.

In the central-scIL-15/Rα format, one preferred embodiment utilizes theskew variant pair S364K/E357Q:L368D/K370S. In one embodiment, thetargeted IL-15/IL-15Rα heterodimeric protein is an“central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and d) a third and fourth monomerthat each include from N-to C-terminus, a VL-CL, where the VH of thefirst monomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, and where the first andsecond variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In an exemplary embodiment, the first variantFc domain includes skew variants L368D and K370S, and the second variantFc domain includes skew variants S364K and E357Q.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and d) a third and fourth monomerthat each include from N-to C-terminus, a VL-CL, where the VH of thefirst monomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D, and where the first and second variant Fc domainsinclude the skew variant pair S364K/E357Q:L368D/K370S. In an exemplaryembodiment, the first variant Fc domain includes skew variants L368D andK370S, and the second variant Fc domain includes skew variants S364K andE357Q. In an exemplary embodiment, the VH and VL are the VH and VL ofany of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S. In one embodiment, the targeted IL-15/IL-15Rαheterodimeric protein is an “central-scIL-15/Rα”format heterodimericprotein that includes: a) a first monomer that includes, from N- toC-terminus, a VH-(domain linker)-IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant-(domain linker)-CH2-CH3, where CH2-CH3 is a firstvariant Fc domain; b) a second monomer that includes, from N- toC-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a second variant Fcdomain; and d) a third and fourth monomer that each include from N-toC-terminus, a VL-CL, where the VH of the first monomer and the VL of thethird monomer are the variable heavy domain and variable light domain of7G8_H3.30_L1.34, respectively, where the VH of the second monomer andthe VL of the fourth monomer are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34, respectively, where the IL-15 variantand the IL-15Rα(sushi) domain form an IL-15 complex, and where the firstand second variant Fc domains include the skew variant pairS364K/E357Q:L368D/K370S. In another embodiment, the targetedIL-15/IL-15Rα heterodimeric protein is an “central-scIL-15/Rα”formatheterodimeric protein that includes: a) a first monomer that includes,from N- to C-terminus, a VH-(domain linker)-IL-15Rα(sushi)domain-(domain linker)-IL-15 variant-(domain linker)-CH2-CH3, whereCH2-CH3 is a first variant Fc domain; b) a second monomer that includes,from N- to C-terminus, a VH-hinge-CH2-CH3, where CH2-CH3 is a secondvariant Fc domain; and d) a third and fourth monomer that each includefrom N-to C-terminus, a VL-CL, where the VH of the first monomer and theVL of the third monomer are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142, respectively, where the VH of the secondmonomer and the VL of the fourth monomer are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142, respectively, where theIL-15 variant and the IL-15Rα(sushi) domain form an IL-15 complex, andwhere the first and second variant Fc domains include the skew variantpair S364K/E357Q:L368D/K370S. In an exemplary embodiment, the firstvariant Fc domain includes skew variants L368D and K370S, and the secondvariant Fc domain includes skew variants S364K and E357Q.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or the variable heavy and light domain pair of2A11_H1.144_L2.142 as shown in FIG. 12 and the skew variant pairS364K/E357Q:L368D/K370S with either the IL-15 N4D/N65D variant or theIL-15 D30N/N65D variant or the IL-15 D30N/E64Q/N65D variant. In oneembodiment, the targeted IL-15/IL-15Rα heterodimeric protein is an“central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3, whereCH2-CH3 is a second variant Fc domain; and d) a third and fourth monomerthat each include from N-to C-terminus, a VL-CL, where the VH of thefirst monomer and the VL of the third monomer form a first LAG-3 bindingdomain, where the VH of the second monomer and the VL of the fourthmonomer form a second LAG-3 binding domain, where the IL-15 variant andthe IL-15Rα(sushi) domain form an IL-15 complex, where VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142, where the IL-15 variant includes amino acidsubstitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D, and where thefirst and second variant Fc domains include the skew variant pairL368D/K370S:S364K/E357Q. In an exemplary embodiment, the first variantFc domain includes skew variants L368D/K370S, and the second variant Fcdomain includes skew variants S364K/E357Q. In a particular embodiment,the IL-15 variant includes amino acid substitutions N4D/N65D and VH andVL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions N4D/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In one embodiment, theIL-15 variant includes amino acid substitutions D30N/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/N65D and VH and VL are the variable heavy domainand variable light domain of 2A11_H1.144_L2.142. In yet anotherembodiment, the IL-15 variant includes amino acid substitutionsD30N/E64Q/N65D and VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34. In another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of2A11_H1.144_L2.142.

In the central-scIL-15/Rα format, one preferred embodiment utilizes theskew variant set S364K/E357Q:L368D/K370S, the pI variantsQ295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a second variant Fc domain; and d) a third and fourthmonomer that each include from N-to C-terminus, a VL-CL, where the VH ofthe first monomer and the VL of the third monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where thefirst variant Fc domain includes skew variants L368D/K370S and thesecond variant Fc domain includes skew variants S364K/E357Q where thefirst and second variant Fc domains each include FcKO variantsE233P/L234V/L235A/G236del/S267K, where the first variant Fc domainincludes pI variants Q295E/N384D/Q418E/N421D, and where numbering isaccording to EU numbering. In an exemplary embodiment, the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the VH and VL are the VH andVL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variantincludes amino acid substitutions N4D/N65D. In another exemplaryembodiment, the VH and VL are the VH and VL of any of the LAG-3 ABDs inFIGS. 12 and 13A-C and the IL-15 variant includes amino acidsubstitutions D30N/N65D. In yet another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/E64Q/N65D.

In the central-scIL-15/Rα format, one preferred embodiment utilizes ananti-LAG-3 ABD having the variable heavy and light domain pair of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 as shown in FIG. 12 with the FIG.21K format, the skew variant set S364K/E357Q:L368D/K370S, the pIvariants Q295E/N384D/Q418E/N421D, the ablation variantsE233P/L234V/L235A/G236_/S267K on both first and second monomers, andoptionally the 428L/434S variants on both first and second monomers.

In one embodiment, the targeted IL-15/IL-15Rα heterodimeric protein isan “central-scIL-15/Rα”format heterodimeric protein that includes: a) afirst monomer that includes, from N- to C-terminus, a VH-(domainlinker)-IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) asecond monomer that includes, from N- to C-terminus, a VH-hinge-CH2-CH3,where CH2-CH3 is a second variant Fc domain; and d) a third and fourthmonomer that each include from N-to C-terminus, a VL-CL, where the VH ofthe first monomer and the VL of the third monomer form a first LAG-3binding domain, where the VH of the second monomer and the VL of thefourth monomer form a second LAG-3 binding domain, where the IL-15variant and the IL-15Rα(sushi) domain form an IL-15 complex, where VHand VL are the variable heavy domain and variable light domain of7G8_H3.30_L1.34 or 2A11_H1.144_L2.142, where the first variant Fc domainincludes skew variants L368D/K370S and the second variant Fc domainincludes skew variants S364K/E357Q where the first and second variant Fcdomains each include FcKO variants E233P/L234V/L235A/G236del/S267K,where the first variant Fc domain includes pI variantsQ295E/N384D/Q418E/N421D, and where numbering is according to EUnumbering. In certain embodiments, the hinge of the first monomerfurther includes variant C220S. In certain embodiments, the first andsecond variant Fc domains each further include half-life extensionvariants M428L/N434S. In a particular embodiment, the IL-15 variantincludes amino acid substitutions N4D/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsN4D/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In one embodiment, the IL-15 variantincludes amino acid substitutions D30N/N65D and VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34. Inanother embodiment, the IL-15 variant includes amino acid substitutionsD30N/N65D and VH and VL are the variable heavy domain and variable lightdomain of 2A11_H1.144_L2.142. In yet another embodiment, the IL-15variant includes amino acid substitutions D30N/E64Q/N65D and VH and VLare the variable heavy domain and variable light domain of7G8_H3.30_L1.34. In another embodiment, the IL-15 variant includes aminoacid substitutions D30N/E64Q/N65D and VH and VL are the variable heavydomain and variable light domain of 2A11_H1.144_L2.142.

V. PARTICULARLY USEFUL EMBODIMENTS OF THE INVENTION

The present invention provides a targeted IL-15/IL-15Rα heterodimericprotein comprising at least two monomers, one of which contains ananti-LAG-3 ABD and the other that contains an IL-15/RA complex, joinedusing heterodimeric Fc domains.

In some embodiments, the first and the second Fc domains have a set ofamino acid substitutions selected from the group consisting ofS267K/L368D/K370S:S267K/S364K/E357Q; S364K/E357Q:L368D/K370S;L368D/K370S:S364K; L368E/K370S:S364K; T411E/K360E/Q362E:D401K;L368D/K370S:S364K/E357L and K370S:S364K/E357Q, according to EUnumbering.

In some instances, the first and/or the second Fc domains have anadditional set of amino acid substitutions comprisingQ295E/N384D/Q418E/N421D, according to EU numbering. In some cases, thefirst and/or the second Fc domains have an additional set of amino acidsubstitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S267K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to EU numbering.

In some embodiments, the IL-15 protein has a polypeptide sequenceselected from the group consisting of SEQ ID NO:1 (full-length humanIL-15) and SEQ ID NO:2 (truncated human IL-15), and the IL-15Rα proteinhas a polypeptide sequence selected from the group consisting of SEQ IDNO:3 (full-length human IL-15Rα) and SEQ ID NO:4 (sushi domain of humanIL-15Rα).

In embodiments the IL-15 protein and the IL-15Rα protein can have a setof amino acid substitutions selected from the group consisting ofE87C:D96/P97/C98; E87C:D96/C97/A98; V49C:S40C; L52C:S40C; E89C:K34C;Q48C:G38C; E53C:L42C; C42S:A37C; and L45C:A37C, respectively.

In some embodiments, the IL-15 protein is a variant protein that has asequence selected from FIGS. 19 and FIG. 20 to reduce potency. In someembodiments, the IL-15 protein is a variant protein having one or moreamino acid substitutions at the IL-15:CD132 interface.

In some embodiments, the LAG-3 antigen binding domain comprises ananti-LAG-3 scFv or an anti-LAG-3 Fab. In an exemplary embodiment, theLAG-3 ABD includes the VH and VL of any of the LAG-3 ABDs depicted inFIGS. 12 and 13A-C.

In an exemplary embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,variant here VH is a variable heavy domain and CH2-CH3 is a secondvariant Fc domain, and c) a light chain that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, where VH and VLform a LAG-3 binding domain, where the IL-15 variant is an IL-15N4D/N65D variant, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q, where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where the firstvariant Fc domain includes pI variants Q295E/N384D/Q418E/N421D, andwhere numbering is according to EU numbering. In certain embodiments,the first and second variant Fc domains each further include half-lifeextension variants M428L/N434S. In certain embodiments, the hinge of thefirst monomer includes also includes amino acid substitution C220S andthe first and second variant Fc domains each further include half-lifeextension variants M428L/N434S. In some embodiments, the VH and VL arethe variable heavy domain and variable light domain of any of the LAG-3ABDs in FIG. 12 or 13A-C. In some embodiments, the VH and VL are thevariable heavy domain and variable light domain of 7G8_H3.30_L1.34 or2A11_H1.144_L2.142 (FIG. 12).

In an exemplary embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,variant here VH is a variable heavy domain and CH2-CH3 is a secondvariant Fc domain, and c) a light chain that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, where VH and VLform a LAG-3 binding domain, where the IL-15 variant is an IL-15D30N/N65D variant, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q, where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where the firstvariant Fc domain includes pI variants Q295E/N384D/Q418E/N421D, andwhere numbering is according to EU numbering. In some embodiments, thehinge of the first monomer also includes amino acid substitution C220S.In certain embodiments, the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In certainembodiments, the hinge of the first monomer includes also includes aminoacid substitution C220S and the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In someembodiments, the VH and VL are the variable heavy domain and variablelight domain of any of the LAG-3 ABDs in FIG. 12 or 13A-C. In someembodiments, the VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 (FIG. 12).

In an exemplary embodiment, the targeted IL-15/IL-15Rα heterodimericprotein is an “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(domainlinker)-CH2-CH3, where CH2-CH3 is a first variant Fc domain; b) a secondmonomer that includes, from N- to C-terminus, a VH-CH1-hinge-CH2-CH3,variant here VH is a variable heavy domain and CH2-CH3 is a secondvariant Fc domain, and c) a light chain that includes from, N- toC-terminus, VL-VC, where VL is a variable light domain, where VH and VLform a LAG-3 binding domain, where the IL-15 variant is an IL-15D30N/E64Q/N65D variant, where the first variant Fc domain includes skewvariants L368D/K370S and the second variant Fc domain includes skewvariants S364K/E357Q, where the first and second variant Fc domains eachinclude FcKO variants E233P/L234V/L235A/G236del/S267K, where the firstvariant Fc domain includes pI variants Q295E/N384D/Q418E/N421D, andwhere numbering is according to EU numbering. In some embodiments, thehinge of the first monomer also includes amino acid substitution C220S.In certain embodiments, the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In certainembodiments, the hinge of the first monomer includes also includes aminoacid substitution C220S and the first and second variant Fc domains eachfurther include half-life extension variants M428L/N434S. In someembodiments, the VH and VL are the variable heavy domain and variablelight domain of any of the LAG-3 ABDs in FIG. 12 or 13A-C. In someembodiments, the VH and VL are the variable heavy domain and variablelight domain of 7G8_H3.30_L1.34 or 2A11_H1.144_L2.142 (FIG. 12).

Useful “backbone” sequences that can be included in the “scIL-15/Rα XFab” format heterodimeric protein are depicted in FIG. 10. In someembodiments, the “scIL-15/Rα X Fab” format heterodimeric protein thatincludes: a) a first monomer that includes, from N- to C-terminus, anIL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where hinge-CH2-CH3 has the amino acid sequence of Chain 2 of “Backbone1” in FIG. 10 (SEQ ID NO: 58); b) a second monomer that includes, fromN- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavydomain and CH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of“Backbone 1” in FIG. 10 (SEQ ID NO: 57), and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain and VC has the sequence of “Constant Light Chain—Kappa” in FIG.11 (SEQ ID NO: 63). In certain embodiments, the “scIL-15/Rα X Fab”format heterodimeric protein that includes: a) a first monomer thatincludes, from N- to C-terminus, an IL-15Rα(sushi) domain-(domainlinker)-IL-15 variant-(hinge)-CH2-CH3, where hinge-CH2-CH3 has the aminoacid sequence of Chain 2 of “Backbone 2” in FIG. 10 (SEQ ID NO: 60); b)a second monomer that includes, from N- to C-terminus, aVH-CH1-hinge-CH2-CH3, where VH is a variable heavy domain andCH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of “Backbone 2”in FIG. 10 (SEQ ID NO: 59), and c) a light chain that includes from, N-to C-terminus, VL-VC, where VL is a variable light domain and VC has thesequence of “Constant Light Chain—Kappa” in FIG. 11 (SEQ ID NO: 63). Insome embodiments, the “scIL-15/Rα X Fab” format heterodimeric proteinthat includes: a) a first monomer that includes, from N- to C-terminus,an IL-15Rα(sushi) domain-(domain linker)-IL-15 variant-(hinge)-CH2-CH3,where hinge-CH2-CH3 has the amino acid sequence of Chain 2 of “Backbone3” in FIG. 10 (SEQ ID NO: 62); b) a second monomer that includes, fromN- to C-terminus, a VH-CH1-hinge-CH2-CH3, where VH is a variable heavydomain and CH1-hinge-CH2-CH3 has the amino acid sequence of Chain 1 of“Backbone 3” in FIG. 10 (SEQ ID NO: 61), and c) a light chain thatincludes from, N- to C-terminus, VL-VC, where VL is a variable lightdomain and VC has the sequence of “Constant Light Chain—Kappa” in FIG.11 (SEQ ID NO: 63). In an exemplary embodiment, the IL-15 variantincludes amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D. In an exemplary embodiment, the IL-15 variant includesamino acid substitutions N4D/N65D, D30N/N65D, or D30N/E64Q/N65D. In anexemplary embodiment, the VH and VL are the VH and VL of any of theLAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includes aminoacid substitutions N4D/N65D. In another exemplary embodiment, the VH andVL are the VH and VL of any of the LAG-3 ABDs in FIGS. 12 and 13A-C andthe IL-15 variant includes amino acid substitutions D30N/N65D. In yetanother exemplary embodiment, the VH and VL are the VH and VL of any ofthe LAG-3 ABDs in FIGS. 12 and 13A-C and the IL-15 variant includesamino acid substitutions D30N/E64Q/N65D.

Particular preferred LAG-3 targeted IL-15/IL-15Rα-Fc heterodimericfusion proteins include XENP27972, XENP27973, XENP27977, XENP27978,XENP029486, XENP029487, XENC1000, XENC1001, XENC1002, XENC1003, XENC1004and XENC1005 “scIL-15/Rα X Fab” format heterodimeric protein. Exemplaryembodiments of the LAG-3 targeted IL-15/IL-15Rα-Fc heterodimeric fusionproteins are shown in FIGS. 22A and B, FIGS. 29A and B, FIGS. 46A and B,FIGS. 47A and B, and FIGS. 48A-D.

VI. NUCLEIC ACIDS OF THE INVENTION

In another aspect, provided herein are nucleic acid compositionsencoding the targeted heterodimeric fusion proteins (or, in the case ofa monomer Fc domain protein, nucleic acids encoding those as well).

As will be appreciated by those in the art, the nucleic acidcompositions will depend on the format of the targeted heterodimericfusion protein. Thus, for example, when the format requires three aminoacid sequences, three nucleic acid sequences can be incorporated intoone or more expression vectors for expression. Similarly, some formatsonly two nucleic acids are needed; again, they can be put into one ortwo expression vectors, or four or 5. As noted herein, some constructshave two copies of a light chain, for example.

As is known in the art, the nucleic acids encoding the components of theinvention can be incorporated into expression vectors as is known in theart, and depending on the host cells used to produce the targetedheterodimeric fusion proteins of the invention. Generally the nucleicacids are operably linked to any number of regulatory elements(promoters, origin of replication, selectable markers, ribosomal bindingsites, inducers, etc.). The expression vectors can be extra-chromosomalor integrating vectors.

The nucleic acids and/or expression vectors provided herein are thentransformed into any number of different types of host cells as is wellknown in the art, including mammalian, bacterial, yeast, insect and/orfungal cells, with mammalian cells (e.g., CHO cells), finding use inmany embodiments.

In some embodiments, nucleic acids encoding each monomer, as applicabledepending on the format, are each contained within a single expressionvector, generally under different or the same promoter controls. Inembodiments of particular use in the present invention, each of thesetwo or three nucleic acids are contained on a different expressionvector.

The targeted heterodimeric fusion protein of the invention are made byculturing host cells comprising the expression vector(s) as is wellknown in the art. Once produced, traditional fusion protein or antibodypurification steps are done, including an ion exchange chromatographystep. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.That is, the inclusion of pI substitutions that alter the isoelectricpoint (pI) of each monomer so that such that each monomer has adifferent pI and the heterodimer also has a distinct pI, thusfacilitating isoelectric purification of the heterodimer (e.g., anionicexchange columns, cationic exchange columns). These substitutions alsoaid in the determination and monitoring of any contaminating homodimerspost-purification (e.g., IEF gels, cIEF, and analytical IEX columns).

VII. BIOLOGICAL AND BIOCHEMICAL FUNCTIONALITY OF TARGETED LAG-3 ANTIBODYX IL-15/IL-15Rα HETERODIMERIC IMMUNOMODULATORY FUSION PROTEINS

Generally the targeted heterodimeric fusion proteins of the inventionare administered to patients with cancer, and efficacy is assessed, in anumber of ways as described herein. Thus, while standard assays ofefficacy can be run, such as cancer load, size of tumor, evaluation ofpresence or extent of metastasis, etc., immuno-oncology treatments canbe assessed on the basis of immune status evaluations as well. This canbe done in a number of ways, including both in vitro and in vivo assays.For example, evaluation of changes in immune status along with “oldfashioned” measurements such as tumor burden, size, invasiveness, LNinvolvement, metastasis, etc. can be done. Thus, any or all of thefollowing can be evaluated: the inhibitory effects of the heterodimericproteins on CD4⁺ T cell activation or proliferation, CD8⁺ T (CTL) cellactivation or proliferation, CD8⁺ T cell-mediated cytotoxic activityand/or CTL mediated cell depletion, NK cell activity and NK mediatedcell depletion, the potentiating effects of the heterodimeric protein onTreg cell differentiation and proliferation and Treg- or myeloid derivedsuppressor cell (MDSC)-mediated immunosuppression or immune tolerance,and/or the effects of heterodimeric protein on proinflammatory cytokineproduction by immune cells, e.g., IL-2, IFN-γ or TNF-α production by Tor other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and ³H-thymidineincorporation method.

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, andothers.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measure either intracellularlyin culture supernatant using cytokines including, but not limited to,IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 using well knowntechniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

A. Assays to Measure Efficacy

In some embodiments, T cell activation is assessed using a MixedLymphocyte Reaction (MLR) assay as is known in the art. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in immune response as measured for an example byphosphorylation or de-phosphorylation of different factors, or bymeasuring other post translational modifications. An increase inactivity indicates immunostimulatory activity. Appropriate increases inactivity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in activation of αβ and/or γδ T cells as measured for anexample by cytokine secretion or by proliferation or by changes inexpression of activation markers like for an example CD137, CD107a, PD1,etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in cytotoxic T cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in NK and/or NKT cell activity as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by changes in expression of activation markerslike for an example CD107a, etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T-cell suppression, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in pro-inflammatory cytokine secretion as measured for exampleby ELISA or by Luminex or by Multiplex bead based methods or byintracellular staining and FACS analysis or by Alispot etc. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in IL-2 secretion as measured for example by ELISA or byLuminex or by Multiplex bead based methods or by intracellular stainingand FACS analysis or by Alispot etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in interferon-y production as measured for example by ELISA orby Luminex or by Multiplex bead based methods or by intracellularstaining and FACS analysis or by Alispot etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th1 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in Th2 response as measured for an example by cytokinesecretion or by changes in expression of activation markers. An increasein activity indicates immunostimulatory activity. Appropriate increasesin activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases cell number and/or activity of at least one of regulatory Tcells (Tregs), as measured for example by flow cytometry or by IHC. Adecrease in response indicates immunostimulatory activity. Appropriatedecreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophages cell numbers, as measured for example byflow cytometry or by IHC. A decrease in response indicatesimmunostimulatory activity. Appropriate decreases are the same as forincreases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in M2 macrophage pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils increase, as measured for example by flowcytometry or by IHC. A decrease in response indicates immunostimulatoryactivity. Appropriate decreases are the same as for increases, outlinedbelow.

In one embodiment, the signaling pathway assay measures increases ordecreases in N2 neutrophils pro-tumorigenic activity, as measured for anexample by cytokine secretion or by changes in expression of activationmarkers. A decrease in response indicates immunostimulatory activity.Appropriate decreases are the same as for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of T cell activation, as measured for an exampleby cytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in inhibition of CTL activation as measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in αβ and/or γδ T cell exhaustion as measured for an exampleby changes in expression of activation markers. A decrease in responseindicates immunostimulatory activity. Appropriate decreases are the sameas for increases, outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases αβ and/or γδ T cell response as measured for an example bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of antigen-specific memory responses asmeasured for an example by cytokine secretion or by proliferation or bychanges in expression of activation markers like for an example CD45RA,CCR7 etc. An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in apoptosis or lysis of cancer cells as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in stimulation of cytotoxic or cytostatic effect on cancercells, as measured for an example by cytotoxicity assays such as for anexample MTT, Cr release, Calcine AM, or by flow cytometry based assayslike for an example CFSE dilution or propidium iodide staining etc. Anincrease in activity indicates immunostimulatory activity. Appropriateincreases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases direct killing of cancer cells as measured for an example bycytotoxicity assays such as for an example MTT, Cr release, Calcine AM,or by flow cytometry based assays like for an example CFSE dilution orpropidium iodide staining etc. An increase in activity indicatesimmunostimulatory activity. Appropriate increases in activity areoutlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases Th17 activity as measured for an example by cytokine secretionor by proliferation or by changes in expression of activation markers.An increase in activity indicates immunostimulatory activity.Appropriate increases in activity are outlined below.

In one embodiment, the signaling pathway assay measures increases ordecreases in induction of complement dependent cytotoxicity and/orantibody dependent cell-mediated cytotoxicity, as measured for anexample by cytotoxicity assays such as for an example MTT, Cr release,Calcine AM, or by flow cytometry based assays like for an example CFSEdilution or propidium iodide staining etc. An increase in activityindicates immunostimulatory activity. Appropriate increases in activityare outlined below.

In one embodiment, T cell activation is measured for an example bydirect killing of target cells like for an example cancer cells or bycytokine secretion or by proliferation or by changes in expression ofactivation markers like for an example CD137, CD107a, PD1, etc. ForT-cells, increases in proliferation, cell surface markers of activation(e.g., CD25, CD69, CD137, PD1), cytotoxicity (ability to kill targetcells), and cytokine production (e.g., IL-2, IL-4, IL-6, IFN-γ, TNF-α,IL-10, IL-17A) would be indicative of immune modulation that would beconsistent with enhanced killing of cancer cells.

In one embodiment, NK cell activation is measured for example by directkilling of target cells like for an example cancer cells or by cytokinesecretion or by changes in expression of activation markers like for anexample CD107a, etc. For NK cells, increases in proliferation,cytotoxicity (ability to kill target cells and increases CD107a,granzyme, and perforin expression), cytokine production (e.g., IFNγ andTNF), and cell surface receptor expression (e.g., CD25) would beindicative of immune modulation that would be consistent with enhancedkilling of cancer cells.

In one embodiment, γδ T cell activation is measured for example bycytokine secretion or by proliferation or by changes in expression ofactivation markers.

In one embodiment, Th1 cell activation is measured for example bycytokine secretion or by changes in expression of activation markers.

Appropriate increases in activity or response (or decreases, asappropriate as outlined above), are increases of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal ineither a reference sample or in control samples, for example testsamples that do not contain a heterodimeric protein of the invention.Similarly, increases of at least one-, two-, three-, four- or five-foldas compared to reference or control samples show efficacy.

VIII. TREATMENTS

Once made, the compositions of the invention find use in a number ofoncology applications, by treating cancer, generally by promoting T cellactivation (e.g., T cells are no longer suppressed) with the binding ofthe heterodimeric fusion proteins of the invention.

Accordingly, the targeted heterodimeric compositions of the inventionfind use in the treatment of these cancers.

A. Targeted Heterodimeric Protein Compositions for In VivoAdministration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (as generally outlined inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, buffers, excipients, or stabilizers are nontoxic to recipientsat the dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

B. Combination Therapies

In some embodiments, the heterodimeric proteins of the invention can beused in combination therapies with antibodies that bind to differentcheckpoint proteins, e.g., not LAG-3 antibodies. In this way, theantigen binding domains of the additional antibody do not compete forbinding with the targeted heterodimeric protein. In this way, a sort of“triple combination” therapy is achieved, as three receptors are engaged(two from the targeted heterodimeric protein and one from the additionalantibody). As discussed herein, the heterodimeric protein can havedifferent valencies and specifities as outlined herein.

Surprisingly, as shown herein, these combinations can result insynergistic effects when co-administered. In this context,“co-administration” means that the two moieties can be administeredsimultaneously or sequentially. That is, in some cases, the drugs may beadministered simultaneously, although generally this is through the useof two separate IV infusions; that is, the drugs are generally notcombined into a single dosage unit. Alternatively, co-administrationincludes the sequential administration of the two separate drugs, eitherin a single day or separate days (including separate days over time).

1. Anti-PD-1 Antibodies for Use in Co-Administration Therapies

As is known in the art, there are two currently approved anti-PD-1antibodies and many more in clinical testing. Thus, suitable anti-PD-1antibodies for use in combination therapies as outlined herein include,but are not limited to, the two currently FDA approved antibodies,pembrolizumab and nivolizumab, as well as those in clinical testingcurrently, including, but not limited to, tislelizumab, Sym021, REGN2810(developed by Rengeneron), JNJ-63723283 (developed by J and J),SHR-1210, pidilizumab, AMP-224, MEDIo680, PDR001 and CT-001, as well asothers outlined in Liu et al., J. Hemat. & Oncol. (2017)10:136, theantibodies therein expressly incorporated by reference. As above,anti-PD-1 antibodies are used in combination when the targetedheterodimeric proteins of the invention do not have an antigen bindingdomain that binds PD-1.

2. Anti-PD-L1 Antibodies for Use in Co-Administration Therapies

In some embodiments, anti-PD-L1 antibodies are used in combination. Asis known in the art, there are three currently approved anti-PD-L1antibodies and many more in clinical testing. Thus, suitable anti-PD-L1antibodies for use in combination therapies as outlined herein include,but are not limited to, the three currently FDA approved antibodies,atezolizumab, avelumab, durvalumab, as well as those in clinical testingcurrently, including, but not limited to, LY33000054 and CS1001, as wellas others outlined in Liu et al., J. Hemat. & Oncol. (2017)10:136, theantibodies therein expressly incorporated by reference. As above,anti-PD-L1 antibodies are used in combination when the targetedheterodimeric proteins of the invention do not have an antigen bindingdomain that binds PD-L1.

3. Anti-TIM-3 Antibodies for Use in Co-Administration Therapies

In some embodiments, anti-TIM-3 antibodies can be used in combinationwith the targeted heterodimeric proteins of the invention. There areseveral TIM-3 antibodies in clinical development, including MBG453 andTSR-022. As above, anti-TIM-3 antibodies are used in combination whenthe targeted heterodimeric proteins of the invention do not have anantigen binding domain that binds TIM-3.

4. Anti-TIGIT Antibodies for Use in Co-Administration Therapies

In some embodiments, anti-TIGIT antibodies can be used in combinationwith the targeted heterodimeric proteins of the invention. There areseveral TIGIT antibodies in clinical development, BMS-986207, OMP-313M32and MTIG7192A. As above, anti-TIGIT antibodies are used in combinationwhen the targeted heterodimeric proteins of the invention do not have anantigen binding domain that binds TIGIT.

5. Anti-CTLA-4 Antibodies for Use in Co-Administration Therapies

In some embodiments, anti-CTLA-4 antibodies can be used in combinationwith the targeted heterodimeric proteins of the invention. Ipilimumabhas been approved, and there are several more in development, includingCP-675,206 and AGEN-1884. As above, anti-CTLA-4 antibodies are used incombination when the targeted heterodimeric proteins of the invention donot have an antigen binding domain that binds CTLA-4.

C. Administrative Modalities

The targeted heterodimeric proteins and chemotherapeutic agents of theinvention are administered to a subject, in accord with known methods,such as intravenous administration as a bolus or by continuous infusionover a period of time.

D. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the targetedheterodimeric protein used in the present invention depend on thedisease or condition to be treated and may be determined by the personsskilled in the art.

An exemplary, non-limiting range for a therapeutically effective amountof a targeted heterodimeric protein used in the present invention isabout 0.1-100 mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

IX. EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in US PatentPublications 2015/0307629, 2014/0288275 and WO2014/145806, all of whichare expressly incorporated by reference in their entirety andparticularly for the techniques outlined therein. Examples 1 and 2 fromU.S. Ser. No. 62,416, 087, filed on Nov. 1, 2016 are expresslyincorporated by reference in their entirety, including the correspondingfigures. Additionally, U.S. Ser. Nos. 62/408,655, 62/443,465,62/477,926, 15/785,401, 62/416,087 and 15/785,393 are expresslyincorporated by reference in their entirety, and specifically for allthe sequences, Figures and Legends therein.

A. Example 1 Anti-LAG-3 ABDs

Examples of antigen-binding domains which bind LAG-3 were described inin WO2017/218707, the contents are hereby incorporated in its entiretyfor all purposes, and in particular for the LAG-3 ABDs in FIG. 11, thedata in FIG. 18, FIG. 55, FIG. 56, FIG. 63 and SEQ ID NO:s 36819-36962,SEQ ID NO:s 35417-35606, SEQ ID NO:s 25914-32793 and SEQ ID NO:s32794-33002 sequences in the sequence listing.

B. Example 2 LAG-3-Targeted IL-15/Rα-Fc Fusions

Reference is made to WO2018/071919 which describes IL-15/RA-Fc fusionsthat do not contain ABDs as are generally depicted in FIGS. 9A-9G andFIGS. 39A-39D. WO2018/071919 is expressly incorporated by referenceherein, and specifically for all of the sequences, formats, Figures andLegends therein.

2A: Generation of LAG-3-Targeted IL-15/Rα-Fc Fusions

Plasmids coding for IL-15, IL-15Rα sushi domain, or the anti-LAG-3variable regions were constructed by standard gene synthesis, followedby subcloning into a pTT5 expression vector containing Fc fusionpartners (e.g., constant regions as depicted in Figure). Cartoonschematics of illustrative LAG-3-targeted IL-15/Rα-Fc fusions aredepicted in FIG. 21.

The “scIL-15/Rα x scFv” format (FIG. 21A) comprises IL-15Rα(sushi) fusedto IL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with an scFv fusedto the other side of the heterodimeric Fc.

The “scFv x ncIL-15/Rα” format (FIG. 21B) comprises an scFv fused to theN-terminus of a heterodimeric Fc-region, with IL-15Rα(sushi) fused tothe other side of the heterodimeric Fc, while IL-15 is transfectedseparately so that a non-covalent IL-15/Rα complex is formed.

The “scFv x dsIL-15/Rα” format (FIG. 21C) is the same as the “scFv xncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15 are covalentlylinked as a result of engineered cysteines.

The “scIL-15/Rα x Fab” format (FIG. 21D) comprises IL-15Rα(sushi) fusedto IL-15 by a variable length linker (termed “scIL-15/Rα”) which is thenfused to the N-terminus of a heterodimeric Fc-region, with a variableheavy chain (VH) fused to the other side of the heterodimeric Fc, whilea corresponding light chain is transfected separately so as to form aFab with the VH. Sequences for illustrative LAG-3-targeted IL-15/Rα-Fcfusion proteins of this format are depicted in Figure.

The “ncIL-15/Rα x Fab” format (FIG. 21E) comprises a VH fused to theN-terminus of a heterodimeric Fc-region, with IL-15Rα(sushi) fused tothe other side of the heterodimeric Fc, while a corresponding lightchain is transfected separately so as to form a Fab with the VH, andwhile IL-15 is transfected separately so that a non-covalent IL-15/Rαcomplex is formed.

The “dsIL-15/Rα x Fab” format (FIG. 21F) is the same as the “ncIL-15/Rαx Fab” format, but wherein IL-15Rα(sushi) and IL-15 are covalentlylinked as a result of engineered cysteines.

The “mAb-scIL-15/Rα” format (FIG. 21G) comprises VH fused to theN-terminus of a first and a second heterodimeric Fc, with IL-15 is fusedto IL-15Rα(sushi) which is then further fused to the C-terminus of oneof the heterodimeric Fc-region, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs.

The “mAb-ncIL-15/Rα” format (FIG. 21H) comprises VH fused to theN-terminus of a first and a second heterodimeric Fc, with IL-15Rα(sushi)fused to the C-terminus of one of the heterodimeric Fc-region, whilecorresponding light chains are transfected separately so as to form aFabs with the VHs, and while and while IL-15 is transfected separatelyso that a non-covalent IL-15/Rα complex is formed.

The “mAb-dsIL-15/Rα” format (FIG. 21I) is the same as the“mAb-ncIL-15/Rα” format, but wherein IL-15Rα(sushi) and IL-15 arecovalently linked as a result of engineered cysteines.

The “central-IL-15/Rα” format (FIG. 21J) comprises a VH recombinantlyfused to the N-terminus of IL-15 which is then further fused to one sideof a heterodimeric Fc and a VH recombinantly fused to the N-terminus ofIL-15Rα(sushi) which is then further fused to the other side of theheterodimeric Fc, while corresponding light chains are transfectedseparately so as to form a Fabs with the VHs.

The “central-scIL-15/Rα” format (FIG. 21K) comprises a VH fused to theN-terminus of IL-15Rα(sushi) which is fused to IL-15 which is thenfurther fused to one side of a heterodimeric Fc and a VH fused to theother side of the heterodimeric Fc, while corresponding light chains aretransfected separately so as to form a Fabs with the VHs.

2B: LAG-3-Targeted IL-15/Rα-Fc Fusions Enhance GVHD, and CombinesSynergistically with Anti-PD-1 Antibody

Illustrative LAG-3-targeted IL-15/Rα-Fc fusion proteins, XENP27972 andXENP27973 alone or in combination with (a bivalent anti-PD-1 mAb basedon nivolumab with ablated effector function; sequences for which isdepicted in FIG. 23), were evaluated in a Graft-versus-Host Disease(GVHD) model conducted in NSG (NOD-SCID-gamma) immunodeficient mice.When the NSG mice are injected with human PBMCs, the human PBMCs developan autoimmune response against mouse cells. Dosing of NSG mice injectedwith human PBMCs followed with LAG-3-targeted IL-15/Rα-Fc fusionproteins proliferate the engrafted T cells and enhances engraftment.

10 million human PBMCs were engrafted into NSG mice via IV-OSP on Day −1followed by dosing with the indicated test articles at the indicatedconcentrations on Days 0, 7, 14, and 21. Counts of various lymphocytepopulations were performed on Days 6 and 10, data for which are depictedin Figures—. Body weights of mice were measured over time and depictedin Figure as percentage of initial body weight. The data show thatdosing with either XENP27972 or XENP27973 following engraftment withhuman PBMCs enhanced GVHD as indicated by increased T cell (CD8+ andCD4+), NK cell, and CD45+ cell counts as well as decreased body weightin comparison to engraftment with PBMC alone. Notably, both XENP27972and XENP27973 enhanced GVHD to a greater extent than dosing withXENP16432 alone. Additionally, the data show that XENP27972 andXENP27973 combine synergistically with XENP16432 in enhancing GVHD asindicated by the death of all mice by Day 19 following dosing with acombination of XENP27972 and XENP16432, and death of all but one mice byDay 19 following dosing with a combination of XENP27973 and XENP16432.This suggests that, in an immuno-oncology setting, treatment withLAG-3-targeted IL-15/Rα-Fc fusion proteins alone or in combination withcheckpoint blockade antibodies will proliferate tumor-infiltratinglymphocytes and enhance anti-tumor activity.

2C: In Vitro Characterization of LAG-3-Targeted IL-15/Rα-Fc Fusions

The LAG-3-targeted IL-15/Rα-Fc fusions were further characterized in acell proliferation assay. Human PBMCs were stimulated for 48 hours with500 ng/ml plate-bound anti-CD3 (OKT3) and then labeled with CFSE andincubated with the following test articles for 4 days at 37° C.:XENP27972 (LAG-3-targeted IL-15/Rα-Fc fusion based on anti-LAG-3 clone7G8); XENP27973 (LAG-3-targeted IL-15/Rα-Fc fusion based on anti-LAG-3done 2A11); XENP24306 (control untargeted IL-15(D30N/E64Q/N65D)/Rα-Fcfusion having D30N/E64Q/N65D IL-15 variant); and XENP26007 (controlRSV-targeted IL-15/Rα-Fc fusion having N4D/N65D IL-15 variant). Cellswere stained with the following antibodies:, anti-CD8-PerCP-Cy5.5 (SK1),anti-CD3-PE-Cy7 (OKT3), anti-CD45RO-APC-Fire750 (UCHL1),anti-HLA-DR-Alexa700 (L243), anti-CD16-BV605 (3G6), anti-CD56-BV605(HCD56), anti-CD25-BV711 (M-A251), anti-CD45RA-BV785 (HI100),anti-CD4-BUV395 (SK3), and Zombie Aqua-BV510 and analyzed by flow forvarious cell populations.

The proliferation of various T cell and NK cell populations based onCFSE dilution (Zombie Aqua to exclude dead cells) was investigated, datafor which are depicted in FIGS. 30-35. The data show that theLAG-3-targeted IL-15/Rα-Fc fusions, in particular XENP27972, are morepotent in inducing proliferation of both CD8⁺ and CD4⁺ T cells incomparison to untargeted IL-15(D30N/E64Q/N65D)/Rα-Fc fusion (as well ascontrol RSV-targeted IL-15/Rα-Fc fusion), with a preference for CD8+ Tcells. Notably, the LAG-3-targeted IL-15/Rα-Fc fusions preferentiallytargets memory T cells, suggesting that in a clinical setting, theLAG-3-targeted IL-15/Rα-Fc fusions will be selective for activatedtumor-infiltrating lymphocytes in the tumor environment.

The activation of various T cell populations based on expression of CD25(a late stage T cell activation marker) and HLA-DR (another activationmarker) were also investigated, data for which are depicted in FIGS.36-38. The data show that LAG-3-targeted IL-15/Rα-Fc fusions generallyappear more potent in inducing activation of CD8 memory T cellpopulations in comparison to untargeted IL-15(D30N/E64Q/N65D)/Rα-Fcfusion (as well as control RSV-targeted IL-15/Rα-Fc fusion).

C. Example 3 LAG-3-Targeted IL-15/Rα-Fc Fusions with Tuned IL-15 Potency3A: IL-15(D30N/N65D) Variant

In a study investigating the pharmacokinetics of IL-15-Fc potencyvariants with Xtend, cynomolgus monkeys were administered a first singleintravenous (i.v.) dose of XENP22853 (WT IL-15/Rα-heteroFc with Xtend;sequences depicted in FIG. 39), XENP24306(IL-15(D30N/E64Q/N65D)/Rα-heteroFc with Xtend; sequences depicted inFIG. 42), XENP24113 (IL-15(N4D/N65D)/Rα-heteroFc with Xtend; sequencesdepicted in FIG. 40), and XENP24294 (scIL-15(N4D/N65D)/Rα-Fc with Xtend;sequences depicted in FIG. 41) at varying concentrations.

FIG. 43 depicts the serum concentration of the test articles over timefollowing the first dose. As expected, incorporating potency variants inaddition to Xtend substitution (as in XENP24306 and XENP24113) greatlyimproves the pharmacokinetics of IL-15-Fc fusions (in comparison toXENP22583). Unexpectedly, however, IL-15/Rα-heteroFc fusion XENP24113and scIL-15/Rα-Fc fusion XENP24294 (which have the same IL-15(N4D/N65D)potency variant) demonstrated reduced pharmacokinetics in comparison toXENP24306. This suggests that the reduced pharmacokinetics was due tothe particular IL-15 potency variant rather than the format of theIL-15-Fc fusion. While a decrease in pharmacokinetics for XENP24113 andXENP24294 was expected on the basis of previous findings whichdemonstrated that the IL-15-Fc fusions having IL-15(N4D/N65D) varianthad greater in vitro potency than IL-15-Fc fusions having theIL-15(D30N/E64Q/N65D) variant, the decrease in pharmacokinetics wasunexpectedly disproportionate to the increase in potency. Accordingly,identification of alternative IL-15 potency variants for use in theLAG-3-targeted IL-15-Fc fusions of the invention was carried out.

It is noted that IL-15(N4D/N65D) has both its substitutions at the IL-15interface responsible for binding to CD122, while IL-15(D30N/E64Q/N65D)has two substitutions (E64Q and N65D) at IL-15:CD122 interface; and onesubstitution (D30N) at the IL-15 interface responsible for binding toCD132. Accordingly, it is believed that the modification at theIL-15:CD132 interface may contribute to the superior pharmacokineticsobserved for XENP24306. Notably, it was determined that scIL-15/Rα-Fcfusions comprising IL-15(N4D/N65D) variant and IL-15(D30N/N65D) variantdemonstrated very similar potency in vitro, as depicted in FIG. 45. Inview of the above, illustrative LAG-3-targeted IL-15-Fc fusioncomprising the IL-15(D30N/N65D) variants were conceived, sequences forwhich are depicted in FIG. 46. A control RSV-targeted IL-15/Rα-Fc fusionprotein XENP29481 with IL-15(D30N/N65D) variant was also generated,sequences for which are depicted in FIG. 49.

3B: IL-15(D30N/E64Q/N65D) Variant

Although the LAG-3-targeted IL-15/Rα-Fc fusions were designed with theaim to be targeted to the tumor environment via the LAG-3-targeting arm,the cytokine moiety is still capable of signaling before reaching thetumor site and may contribute to systemic toxicity. Accordingly,LAG-3-targeted IL-15/Rα-Fc fusions with IL-15(D30N/E64Q/N65D) variantwere constructed to further reduce the IL-15 potency, which asillustrated in Example 2C has drastically reduced activity and in FIG.45. Sequences for illustrative LAG-3-targeted IL-15/Rα-Fc fusionscomprising IL-15(D30N/E64Q/N65D) variant are depicted in FIG. 47.Additionally, XENP30432, a RSV-targeted IL-15/Rα-Fc fusion comprisingIL-15(D30N/E64Q/N65D) variant (sequences for which are depicted in FIG.49) was constructed, to act as a surrogate for investigating thebehavior of LAG-3-targeted IL-15/Rα-Fc fusions comprisingIL-15(D30N/E64Q/N65D) variant outside of the tumor environment.

What is claimed is:
 1. A heterodimeric fusion protein comprising: a) afirst monomer comprising, from N-to C-terminal: i) an IL-15Rα(sushi)domain; ii) a first domain linker; iii) an IL-15 variant; iv) a hinge;and v) a first variant Fc domain comprising CH2-CH3; and b) a secondmonomer comprising, from N-to C-terminal, VH-CH1-hinge-CH2-CH3, whereinthe CH2-CH3 is a second variant Fc domain; and c) a third monomercomprising a VL-CL, wherein the VH and VL are a variable heavy domainand a variable light domain, respectively, that form a human LAG-3antigen binding domain, wherein the first variant Fc domain comprisesskew variants L368D/K370S and the second variant Fc domain comprisesskew variants S364K/E357Q, wherein the first and second variant Fcdomains each comprise FcKO variants E233P/L234V/L235A/G236del/S267K,wherein the first variant Fc domain comprises pI variantsQ295E/N384D/Q418E/N421D, and wherein numbering is according to EUnumbering.
 2. A heterodimeric fusion protein according to claim 1,wherein the hinge of the first monomer comprises amino acid substitutionC220S, and wherein numbering is according to EU numbering.
 3. Aheterodimeric fusion protein according to claim 1 or 2, wherein thefirst and second variant Fc domains each further comprise half-lifeextension variants
 4. A heterodimeric fusion protein according to anyone of claims 1 to 3, wherein the IL-15 variant comprises an amino acidsubstitution(s) selected from the group consisting of N1D, N4D, D8N,D30N, D61N, E64Q, N65D, Q108E, N4D/N65D, D30N/N65D, and D30N/E64Q/N65D.5. A heterodimeric fusion protein according to claim 4, wherein theIL-15 variant comprises amino acid substitutions N4D/N65D, D30N/N65D, orD30N/E64Q/N65D
 6. A heterodimeric fusion protein according to any one ofclaims 1 to 5, wherein the VH and VL are the variable heavy domain andvariable domain of any of the LAG-3 antigen binding domains in FIGS. 12and
 13. 7. A heterodimeric fusion protein according to claim 6, whereinthe LAG-3 antigen binding domain is selected from 2A11_H1.144_L2.142 and7G8_H3.30_L1.34.
 8. A heterodimeric fusion protein according to claim 1,wherein the heterodimeric fusion protein is selected from the groupconsisting of: XENP27972, XENP27973, XENP27977, XENP27978, XENP029486,XENP029487, XENC1000, XENC1001, XENC1002, XENC1003, XENC1004 andXENC1005.
 9. A nucleic acid composition comprising: a) a first nucleicacid encoding said first monomer of any of claims 1 to 8; b) a secondnucleic acid encoding said second monomer of any of claims 1 to 8; a) athird nucleic acid encoding said third monomer of any of claims 1 to 8;respectively.
 10. An expression vector composition comprising: a) afirst expression vector comprising said first nucleic acid of claim 9;b) a second expression vector comprising said second nucleic acid ofclaim 9; and c) a third expression vector comprising said third nucleicacid of claim
 9. 11. A host cell comprising the expression vectorcomposition according to claim
 10. 12. A method of making aheterodimeric fusion protein comprising culturing the host cell of claim11 and recovering the heterodimeric fusion protein from the cellculture.